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Cell_Biology_Alberts. chromosome becomes covalently attached to another. This can result in chromosomes with two centromeres and chromosomes lacking centromeres altogether; both processing of processing of 5˜ endsDNA ends by nuclease deletion of DNA sequence damage repaired accurately using sister chromatid as the template types of aberrant chromosomes are missegregated during cell division. As previously discussed, the specialized structure of telomeres prevents the natural ends of chromosomes from being mistaken for broken DNA and “repaired” in this way.


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Cell_Biology_Alberts. A much more accurate type of double-strand break repair occurs in newly replicated DNA (Figure 5–45B). Here, the DNA is repaired using the sister chromatid as a template. This reaction is an example of homologous recombination, and we consider its mechanism later in this chapter. Most organisms employ both non-homologous end joining and homologous recombination to repair double-strand breaks in DNA. Nonhomologous end joining predominates in humans; homologous recombination is used only during and shortly after DNA replication (in S and G2 phases), when sister chromatids are available to serve as templates. repaired DNA has generally suffered a deletion of nucleotides (A)


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Cell_Biology_Alberts. repaired DNA has generally suffered a deletion of nucleotides (A) Figure 5–45 Two ways to repair double-strand breaks. (A) Nonhomologous end joining alters the original DNA sequence when repairing a broken chromosome. The initial degradation of the broken DNA ends is important because the nucleotides at the site of the initial break are often damaged and cannot be ligated. Nonhomologous end joining usually takes place when cells have not yet duplicated their DNA. (B) Repairing double-strand breaks by homologous recombination is more difficult to accomplish but restores the original DNA sequence. It typically takes place after the DNA has been duplicated (when a duplex template is available) but before the cell has divided. Details of the homologous recombination pathway are presented in the following section (see Figure 5–48).


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Cell_Biology_Alberts. Figure 5–46 Nonhomologous end joining. (A) A central role is played by the Ku protein, a heterodimer that grasps the broken chromosome ends. The additional proteins shown are needed to hold the broken ends together while they are processed and eventually joined covalently. (B) Three-dimensional structure of a Ku heterodimer bound to the end of a duplex DNA fragment. The Ku protein is also essential for V(D)J joining, a specific recombination process through which antibody and T cell receptor diversity is generated in developing B and T cells (discussed in Chapter 24). V(D)J joining and nonhomologous end joining show many similarities in mechanism but the former relies on specific double-strand breaks produced deliberately by the cell. (B, from J.R. Walker, R.A. Corpina, and J. Goldberg, Nature 412:607–614, 2001. With permission from Macmillan Publishers Ltd.) DNA Damage Delays Progression of the Cell Cycle


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Cell_Biology_Alberts. DNA Damage Delays Progression of the Cell Cycle We have just seen that cells contain multiple enzyme systems that can recognize and repair many types of DNA damage (Movie 5.7). Because of the importance of maintaining intact, undamaged DNA from generation to generation, eukaryotic cells have an additional mechanism that maximizes the effectiveness of their DNA repair enzymes: they delay progression of the cell cycle until DNA repair is complete. As discussed in detail in Chapter 17, the orderly progression of the cell cycle is stopped if damaged DNA is detected, and it restarts when the damage has been repaired. Thus, in mammalian cells, the presence of DNA damage can block entry from G1 into S phase, it can slow S phase once it has begun, and it can block the transition from G2 phase to M phase. These delays facilitate DNA repair by providing the time needed for the repair to reach completion.


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Cell_Biology_Alberts. DNA damage also results in an increased synthesis of some DNA repair enzymes. This response depends on special signaling proteins that sense DNA damage and up-regulate the appropriate DNA repair enzymes. The importance of this mechanism is revealed by the phenotype of humans who are born with defects in the gene that encodes the ATM protein. These individuals have the disease ataxia telangiectasia (AT ), the symptoms of which include neurodegeneration, a predisposition to cancer, and genome instability. The ATM protein is a large kinase needed to generate the intracellular signals that sound the alarm in response to many types of spontaneous DNA damage (see Figure 17–62), and individuals with defects in this protein therefore suffer from the effects of unrepaired DNA lesions.


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Cell_Biology_Alberts. Genetic information can be stored stably in DNA sequences only because a large set of DNA repair enzymes continuously scan the DNA and replace any damaged nucleotides. Most types of DNA repair depend on the presence of a separate copy of the genetic information in each of the two strands of the DNA double helix. An accidental lesion on one strand can therefore be cut out by a repair enzyme and a corrected strand resynthesized by reference to the information in the undamaged strand.


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Cell_Biology_Alberts. Most of the damage to DNA bases is excised by one of two major DNA repair pathways. In base excision repair, the altered base is removed by a DNA glycosylase enzyme, followed by excision of the resulting sugar phosphate. In nucleotide excision repair, a small section of the DNA strand surrounding the damage is removed from the DNA double helix as an oligonucleotide. In both cases, the gap left in the DNA helix is filled in by the sequential action of DNA polymerase and DNA ligase, using the undamaged DNA strand as the template. Some types of DNA damage can be repaired by a different strategy—the direct chemical reversal of the damage— which is carried out by specialized repair proteins. When DNA damage is excessive, a special class of inaccurate DNA polymerases, called translesion polymerases, is used to bypass the damage, allowing the cell to survive but sometimes creating permanent mutations at the sites of damage.


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Cell_Biology_Alberts. Other critical repair systems—based on either nonhomologous end joining or homologous recombination—reseal the accidental double-strand breaks that occur in the DNA helix. In most cells, an elevated level of DNA damage causes a delay in the cell cycle, which ensures that DNA damage is repaired before a cell divides.


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Cell_Biology_Alberts. In the two preceding sections, we discussed the mechanisms that allow the DNA sequences in cells to be maintained from generation to generation with very little change. In this section, we further explore one of the DNA repair mechanisms, a diverse set of reactions known collectively as homologous recombination. The key feature of homologous recombination (also known as general recombination) is an exchange of DNA strands between a pair of homologous duplex DNA sequences, that is, segments of double helix that are very similar or identical in nucleotide sequence. This exchange allows one stretch of duplex DNA to act as a template to restore lost or damaged information on a second stretch of duplex DNA. Because the template for repair is not limited to the strand complementary to that containing the damage, homologous recombination can repair many types of DNA damage. It is, for example, the main way to accurately repair double-strand breaks, as introduced in the previous section (see


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Cell_Biology_Alberts. the damage, homologous recombination can repair many types of DNA damage. It is, for example, the main way to accurately repair double-strand breaks, as introduced in the previous section (see Figure 5–45B). Double-strand breaks can result from radiation and reactive chemicals, but most of the time they arise from DNA replication forks that become stalled or broken independently of any such external cause. Homologous recombination accurately corrects these accidents and, because they occur during nearly every round of DNA replication, this repair mechanism is essential for every proliferating cell. Homologous recombination is perhaps the most versatile DNA repair mechanism available to the cell; the “all-purpose” nature of recombinational repair probably explains why its mechanism and the proteins that carry it out have been conserved in virtually all cells on Earth.


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Cell_Biology_Alberts. Additionally, we shall see that homologous recombination plays a special role in sexually reproducing organisms. During meiosis, a key step in gamete (sperm and egg) production, it catalyzes the orderly exchange of bits of genetic information between corresponding (homologous) maternal and paternal chromosomes to create new combinations of DNA sequences in the chromosomes passed to the offspring.


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Cell_Biology_Alberts. The current view of homologous recombination as a critical DNA repair mechanism in all cells evolved slowly from its original discovery as a key component in the specialized process of meiosis in plants and animals. The subsequent recognition that homologous recombination also occurs in unicellular organisms made it much more amenable to molecular analyses. Thus, most of what we know about the biochemistry of genetic recombination was originally derived from studies of bacteria, especially of E. coli and its viruses, as well as from experiments with simple eukaryotes such as yeasts. For these organisms with short generation times and relatively small genomes, it was possible to isolate a large set of mutants with defects in their recombination processes. The protein altered in each mutant was then identified and, ultimately, studied biochemically. Close relatives of these proteins have been found in more complex eukaryotes including flies, mice, and humans, and more recently, it has


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Cell_Biology_Alberts. was then identified and, ultimately, studied biochemically. Close relatives of these proteins have been found in more complex eukaryotes including flies, mice, and humans, and more recently, it has been possible to directly analyze homologous recombination in these species as well. These studies reveal that the fundamental processes that catalyze homologous recombination are common to all cells.


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Cell_Biology_Alberts. The hallmark of homologous recombination is that it takes place only between DNA duplexes that have extensive regions of sequence similarity (homology). Not surprisingly, base-pairing underlies this requirement, and two DNA duplexes that are undergoing homologous recombination “sample” each other’s DNA sequence by engaging in extensive base-pairing between a single strand from one DNA duplex and the complementary single strand from the other. The match need not be perfect, but it must be very close for homologous recombination to succeed.


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Cell_Biology_Alberts. In its simplest form, this type of base-pairing interaction can be mimicked in a test tube by allowing a DNA double helix to re-form from its separated single strands. This process, called DNA renaturation or hybridization, occurs when a rare random collision juxtaposes complementary nucleotide sequences on two matching DNA single strands, allowing the formation of a short stretch of double helix between them. This relatively slow helix-nucleation step is followed by a very rapid “zippering” step, as the region of double helix is extended to maximize the number of base-pairing interactions (Figure 5–47).


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Cell_Biology_Alberts. DNA hybridization can create a region of DNA double helix consisting of strands that originate from two different duplex DNA molecules as long as they are complementary, or nearly so. As we will see shortly, the formation of such a hybrid molecule, known as a heteroduplex, is an essential feature of homologous recombination. DNA hybridization and heteroduplex formation is also the basis for many of the methods used to study cells, and we will discuss these uses in Chapter 8. The DNA in a living cell is almost all in the stable double-helical form, so the reaction depicted in Figure 5–47 rarely occurs in vivo. Instead, as we shall see, homologous recombination is brought about through a carefully controlled set of reactions that allow two DNA duplexes to sample each other’s sequences without fully dissociating into single strands.


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Cell_Biology_Alberts. We saw in the previous section that nonhomologous end-joining occurs without a template and usually leaves a mutation at the site at which a double-strand break is repaired. In contrast, homologous recombination can repair double-strand breaks accurately, without any loss or alteration of nucleotides at the site of repair. For homologous recombination to do this repair job, the broken DNA has to be brought into proximity with homologous but unbroken DNA, which can serve as a template for repair. For this reason, homologous recombination often occurs just after DNA replication, when the two daughter DNA molecules lie close together and one can serve as a template for repair of the other. As we shall see, the process of DNA replication itself creates a special risk of accidents requiring this sort of repair.


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Cell_Biology_Alberts. The simplest pathway through which homologous recombination can repair double-strand breaks is shown in Figure 5–48. In essence, the broken DNA duplex and the template duplex carry out a “strand dance” so that one of the damaged strands can use the complementary strand of the intact DNA duplex as a template for repair. First, the ends of the broken DNA are chewed back, or “resected,” by specialized nucleases to produce overhanging, single-strand 3ʹ ends. The next step is strand exchange (also called strand invasion), during which one of the single-strand 3ʹ ends from the damaged DNA molecule worms its way into the template duplex and searches it for homologous sequences through base-pairing. We describe this remarkable reaction in detail in the next section. Once stable base-pairing is established (which completes the strand exchange step), an accurate DNA polymerase extends the invading strand by using the information provided by the undamaged template molecule, thus restoring the


Cell_Biology_Alberts

Cell_Biology_Alberts. established (which completes the strand exchange step), an accurate DNA polymerase extends the invading strand by using the information provided by the undamaged template molecule, thus restoring the damaged DNA. The last steps—strand displacement, further repair synthesis, and ligation—restore the two original DNA double helices and complete the repair process. Homologous recombination resembles other DNA repair reactions in that a


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Cell_Biology_Alberts. Figure 5–47 DNA hybridization. DNA double helices can re-form from their separated strands in a reaction that depends on the random collision of two complementary DNA strands. The vast majority of such collisions are not productive, as shown on the left, but a few result in a short region where complementary base pairs have formed (helix nucleation). A rapid zippering then leads to the formation of a complete double helix. Through this trial-and-error process, a DNA strand will find its complementary partner even in the midst of millions of nonmatching DNA strands. DNA polymerase utilizes a pristine template to restore damaged DNA. However, instead of using the partner complementary strand as a template, as occurs in most DNA repair pathways, homologous recombination exploits a complementary strand from a separate DNA duplex. Strand Exchange Is Carried Out by the RecA/Rad51 Protein


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Cell_Biology_Alberts. Strand Exchange Is Carried Out by the RecA/Rad51 Protein Of all the steps of homologous recombination, strand exchange is the most difficult to imagine. How does the invading single strand rapidly sample a DNA duplex for homology? Once the homology is found, how does the exchange occur? How is the inherent stability of the template double helix overcome? The answers to these questions came from biochemical and structural studies of the protein that carries out these feats, called RecA in E. coli and Rad51 in virtually all eukaryotic organisms. To catalyze strand exchange, RecA first binds cooperatively to the invading single strand, forming a protein–DNA filament that forces the DNA into an unusual configuration: groups of three consecutive nucleotides are held as though they were in a conventional DNA double helix but, between adjacent triplets, the DNA backbone is untwisted and stretched out (Figure 5–49). This unusual protein–DNA filament then binds to duplex DNA


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Cell_Biology_Alberts. Figure 5–48 Mechanism of double-strand break repair by homologous recombination. This is the preferred method for repairing DNA double-strand breaks that arise shortly after the DNA has been replicated, while the daughter DNA molecules are still held close together. In general, homologous recombination can be regarded as a flexible series of reactions, with the exact pathway differing from one case to the next. For example, the length of the repair “patch” can vary considerably depending on the extent of 5ʹ processing and new DNA synthesis, indicated in green.


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Cell_Biology_Alberts. in a way that stretches the duplex, destabilizing it and making it easy to pull the strands apart. The invading single strand then can sample the sequence of the duplex by conventional base-pairing. This sampling occurs in triplet nucleotide blocks: if a triplet match is found, the adjacent triplet is sampled, and so on. In this way, mismatches quickly lead to dissociation and only an extended stretch of base-pairing (at least 15 nucleotides) stabilizes the invading strand and leads to strand exchange.


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Cell_Biology_Alberts. RecA hydrolyzes ATP, and the steps described above require that each RecA monomer along the filament be in the ATP-bound state. However, the searching itself does not require ATP hydrolysis; instead, the process occurs by simple molecular collision, allowing many potential sequences to be rapidly sampled. Once the strand-exchange reaction is completed, however, ATP hydrolysis is necessary to disassemble RecA from the complex of DNA molecules. At this point, repair DNA polymerases and DNA ligase can complete the repair process, as shown in Figure 5–48.


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Cell_Biology_Alberts. Although accurately repairing double-strand breaks, which can arise from radiation or chemical reactions, is a crucial function of homologous recombination, perhaps its most important role is in rescuing stalled or broken DNA replication forks. Many types of events can cause a replication fork to break, and here we consider just one example: a single-strand nick or gap in the parental DNA helix just ahead of a replication fork. When the fork reaches this lesion, it falls apart—resulting in one broken and one intact daughter chromosome. The broken fork can be flawlessly repaired (Figure 5–50) using the same basic homologous recombination reactions we discussed above for the repair of double-strand breaks. With slight modifications, the set of reactions depicted in Figures 5–48 and 5–50— known collectively as homologous recombination—can accurately repair many different types of DNA damage. Cells Carefully Regulate the Use of Homologous Recombination in DNA Repair


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Cell_Biology_Alberts. Cells Carefully Regulate the Use of Homologous Recombination in DNA Repair Although homologous recombination neatly solves the problem of accurately repairing double-strand breaks and other types of DNA damage, it does present


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Cell_Biology_Alberts. Figure 5–49 Strand invasion catalyzed by the RecA protein. Our understanding of this reaction is based in part on structures determined by x-ray diffraction studies of RecA bound to singleand double-strand DNA. These DNA structures (shown without the RecA protein) are on the left side of the diagram. Starting at the top, ATP-bound RecA associates with single-strand DNA, holding it in an elongated form where groups of three bases are separated from each other by a stretched and twisted backbone. In the next step, the RecA-bound single strand then binds to duplex DNA, destabilizing it and allowing the single strand to sample its sequence through base-pairing, three bases at a time. If no match is found, the RecA-bound single strand of DNA rapidly dissociates and begins a new search. If an extensive match is found, the structure is disassembled through ATP hydrolysis, resulting in the dissociation of RecA and the exchange of one single strand of DNA for another, thereby forming a


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Cell_Biology_Alberts. If an extensive match is found, the structure is disassembled through ATP hydrolysis, resulting in the dissociation of RecA and the exchange of one single strand of DNA for another, thereby forming a heteroduplex. (PDB code: 3CMX.)


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Cell_Biology_Alberts. Figure 5–50 Repair of a broken replication fork by homologous recombination. When a moving replication fork encounters a single-strand break, it will collapse, but can be repaired by homologous recombination. The process uses many of the same reactions shown in Figure 5–48 and proceeds through the same basic steps. Green strands represent the new DNA synthesis that takes place after the replication fork has broken. This pathway allows the fork to move past the site that was nicked on the original template by using the undamaged duplex as a template to synthesize DNA. (Adapted from M.M. Cox, Proc. Natl Acad. Sci. USA 98:8173–8180, 2001. With permission from National Academy of Sciences.) some dangers to the cell as it sometimes “repairs” damage using the wrong bit of the genome as the template. For example, sometimes a broken human chromosome is “repaired” using the homolog from the other parent instead of the sister chromatid as the template. Because maternal and paternal chromosomes


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Cell_Biology_Alberts. For example, sometimes a broken human chromosome is “repaired” using the homolog from the other parent instead of the sister chromatid as the template. Because maternal and paternal chromosomes differ in DNA sequence at many positions along their lengths, this type of repair can convert the sequence of the repaired DNA from the maternal to the paternal sequence or vice versa. The result of this type of errant recombination is known as loss of heterozygosity. It can have severe consequences if the homolog used for repair contains a deleterious mutation, because the recombination event destroys the “good” copy. Loss of heterozygosity, although rare, is a critical step in the formation of many cancers (discussed in Chapter 20).


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Cell_Biology_Alberts. Cells go to great lengths to minimize the risk of mishaps of these types; indeed, nearly every step of homologous recombination is carefully regulated. For example, the first step, processing of the broken ends, is coordinated with the cell cycle: the nuclease enzymes that carry out this process are activated (in part, by phosphorylation) only in the S and G2 phases of the cell cycle, when a daughter duplex (either as a partially replicated chromosome or a fully replicated sister chromatid) can serve as a template for repair (see Figure 5–50). The close proximity of the two daughter chromosomes disfavors the use of other genome sequences in the repair process.


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Cell_Biology_Alberts. The loading of RecA or Rad52 onto the processed DNA ends and the subsequent strand-exchange reaction are also tightly controlled. Although these proteins alone can carry out these steps in vitro, a series of accessory proteins, including Rad52, is needed in eukaryotic cells to ensure that homologous recombination is efficient and accurate (Figure 5–51). There are many such accessory proteins, and exactly how they coordinate and control homologous recombination remains a mystery. We do know that the enzymes that catalyze recombinational repair are made at relatively high levels in eukaryotes and are dispersed throughout the nucleus in an inactive form. In response to DNA damage, they rapidly converge on the sites of DNA damage, become activated, and form “repair factories” where many lesions are apparently brought together and repaired (Figure 5–52).


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Cell_Biology_Alberts. In Chapter 20, we shall see that both too much and too little homologous recombination can lead to cancer in humans, the former through repair using the “wrong” template (as described above) and the latter through an increased mutation rate caused by inefficient DNA repair. Clearly, a delicate balance has evolved that keeps this process in check on undamaged DNA, while still allowing it to act efficiently and rapidly on DNA lesions as soon as they arise. Not surprisingly, mutations in the components that carry out and regulate homologous recombination are responsible for several inherited forms of cancer. Two of these, the Brca1 and Brca2 proteins, were first discovered because


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Cell_Biology_Alberts. Figure 5–51 Structure of a portion of the Rad52 protein. This doughnutshaped structure is composed of 11 subunits. Single-strand DNA has been modeled into the deep groove running along the protein surface. Rad52 helps load Rad51 onto single-strand DNA to form the nucleoprotein filament that carries out strand exchange. Rad52 also acts later to re-form the double helix and complete the homologous recombination reaction. (From


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Cell_Biology_Alberts. M.R. Singleton et al., Proc. Natl Acad. Sci. USA 99:13492–13497, 2002. With permission from National Academy of Sciences.) mutations in their genes lead to a greatly increased frequency of breast cancer. Because these mutations cause inefficient repair by homologous recombination, accumulation of DNA damage can, in a small proportion of cells, give rise to a cancer. Brca1 regulates an early step in broken-end processing; without it, such ends are not processed correctly for homologous recombination and instead are repaired inaccurately by the nonhomologous end-joining pathway (see Figure 5–45). Brca2 binds to the Rad51 protein, preventing its polymerization on DNA, and thereby maintaining it in an inactive form until it is needed. Normally, upon DNA damage, Brca2 helps to bring Rad51 protein rapidly to sites of damage and, once in place, to release it in its active form onto single-strand DNA.


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Cell_Biology_Alberts. We have seen that homologous recombination comprises a group of reactions— including broken-end processing, strand exchange, limited DNA synthesis, and ligation—to exchange DNA sequences between two double helices of similar nucleotide sequence. Having discussed its role in accurately repairing damaged DNA, we now turn to homologous recombination as a means to generate DNA molecules that carry novel combinations of genes as a result of the deliberate exchange of material between different chromosomes. Although this occasionally occurs by accident in mitotic cells (and is often detrimental), it is a frequent and necessary part of meiosis, which occurs in sexually reproducing organisms such as fungi, plants, and animals.


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Cell_Biology_Alberts. Here, homologous recombination occurs as an integral part of the process whereby chromosomes are parceled out to germ cells (sperm and eggs in animals). We discuss the process of meiosis in detail in Chapter 17; in the following sections, we discuss how homologous recombination during meiosis produces chromosome crossing-over and gene conversion, resulting in hybrid chromosomes that contain genetic information from both the maternal and paternal homologs (Figure 5–53). Crossing-over and gene conversion are both generated by homologous recombination mechanisms that, at their core, resemble those used to repair double-strand breaks. Meiotic Recombination Begins with a Programmed Double-Strand Break


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Cell_Biology_Alberts. Meiotic Recombination Begins with a Programmed Double-Strand Break Homologous recombination in meiosis starts with a bold stroke: a specialized protein (called Spo11 in budding yeast) breaks both strands of the DNA double helix in one of the recombining chromosomes (Figure 5–54). Like a topoisomerase, Spo11, after catalyzing this reaction, remains covalently bound to the broken site of gene site of conversion crossover


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Cell_Biology_Alberts. Figure 5–52 Experiment demonstrating the rapid localization of repair proteins to DNA double-strand breaks. Human fibroblasts were x-irradiated to produce DNA double-strand breaks. Before the x-rays struck the cells, they were passed through a microscopic grid with x-ray-absorbing “bars” spaced 1 μm apart. This produced a striped pattern of DNA damage, allowing a comparison of damaged and undamaged DNA in the same nucleus. (A) Total DNA in a fibroblast nucleus stained with the dye DAPI. (B) Sites of new DNA synthesis due to repair of DNA damage, indicated by incorporation of BudR (a thymidine analog) and subsequent staining with fluorescently labeled antibodies to BudR (green).


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Cell_Biology_Alberts. (C) Localization of the Mre11 complex to damaged DNA as visualized by antibodies against the Mre11 subunit (red). Mre11 is a nuclease that processes damaged DNA in preparation for homologous recombination (see Figure 5–48). (A), (B), and (C) were processed 30 minutes after x-irradiation. (From B.E. Nelms et al., Science 280:590– 592, 1998. With permission from AAAS.) Figure 5–53 Chromosome crossing-over occurs in meiosis. Meiosis is the process by which a diploid cell gives rise to four haploid germ cells, as described in detail in Chapter 17. Meiosis produces germ cells in which the paternal and maternal genetic information (red and blue) has been reassorted through chromosome crossovers. In addition, many short regions of gene conversion occur, as indicated.


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Cell_Biology_Alberts. Figure 5–54 Homologous recombination during meiosis can generate chromosome crossovers. Once the meiosis-specific protein Spo11 and the Mre11 complex break the duplex DNA and process the ends, homologous recombination can proceed along alternative pathways. One (right side of figure) closely resembles the double-strand break repair reaction shown in Figure 5–48 and results in chromosomes that have been “repaired” but have not crossed over. The other (left side with strand breaks as shown by the blue arrows) proceeds through a double Holliday junction and produces two chromosomes that have crossed over. During meiosis, homologous recombination takes place between maternal and paternal chromosome homologs when they are held tightly together (see Figure 17–54). DNA (see Figure 5–21). A specialized nuclease then rapidly degrades the ends bound by Spo11, removing the protein along with the DNA and leaving protruding 3ʹ single-strand ends.


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Cell_Biology_Alberts. DNA (see Figure 5–21). A specialized nuclease then rapidly degrades the ends bound by Spo11, removing the protein along with the DNA and leaving protruding 3ʹ single-strand ends. At this point, many of the recombination reactions resemble those described above for the repair of double-strand breaks; indeed, some of the same proteins are used for both processes. However, several meiosis-specific proteins direct them to perform their tasks somewhat differently, resulting in the distinctive outcomes observed for meiosis. Another important difference is that, in meiosis, recombination occurs preferentially between maternal and paternal chromosomal homo-logs rather than between the newly replicated, identical DNA duplexes that pair in double-strand break repair. In the sections that follow, we describe in more detail those aspects of homologous recombination that are especially important for meiosis.


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Cell_Biology_Alberts. Of special importance in meiosis is an intermediate known as a Holliday junction or cross-strand exchange (Figure 5–55). Each Holliday junction can adopt multiple conformations and a special set of recombination proteins binds to, and thereby stabilizes, the open, symmetric isomer. Specialized proteins that bind to Holliday junctions can catalyze a reaction known as branch migration (Figure 5–56), whereby DNA is spooled through the Holliday junction by continually breaking and re-forming base pairs (Figure 5–57). In this way, the Holliday junction proteins use ATP hydrolysis to expand the region of heteroduplex DNA initially created by the strand-exchange reaction. In meiosis, heteroduplex regions often “migrate” thousands of nucleotides from the original site of the double-strand break. As shown in Figure 5–54, Holliday junctions usually occur in pairs, known as double Holliday junctions.


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Cell_Biology_Alberts. As shown in Figure 5–54, there are two basic outcomes of homologous recombination during meiosis. In humans, approximately 90% of the double-strand breaks produced during meiosis are resolved as non-crossovers (see right side of Figure 5–54). Here, the two original DNA duplexes separate from each other in a form unaltered except for a region of heteroduplex that formed near the site of the original double-strand break. This set of reactions resembles that described above for the repair of double-strand breaks (see Figure 5–48). The other outcome is more profound: a double Holliday junction is formed and is cleaved by specialized enzymes to create a crossover (see left side of Figure 5–54). The two original portions of each chromosome upstream and downstream


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Cell_Biology_Alberts. Figure 5–55 A Holliday junction. The initially formed structure (A) is usually drawn with two strands crossing, as in Figure 5–54. An isomerization of the Holliday junction produces an open, symmetrical structure that is bound by specialized proteins. These proteins “move” the Holliday junctions by a coordinated set of branch-migration reactions (see Figure 5–57 and Movie 5.8). (D) Structure of the Holliday junction in the open form depicted in (B). The Holliday junction is named for the scientist who first proposed its formation. (PDB code: 1DCW.) direction of branch migration


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Cell_Biology_Alberts. Figure 5–56 Simplified view of branch migration. In branch migration, base pairs are continually broken and formed as the branch point moves. Although branch migration can happen spontaneously on naked DNA molecules, the process is inefficient and the branch moves back and forth at random. In the cell, branch migration is carried out using specialized proteins and ATP hydrolysis to ensure that, as shown, the branch moves rapidly and in one direction. As shown in Figure 5–57, branch migrations often occur at Holliday junctions, where two branch-migration reactions are coupled.


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Cell_Biology_Alberts. Figure 5–57 Enzyme-catalyzed branch movement at a Holliday junction by branch migration. In E. coli, a tetramer of the RuvA protein (green) and two hexamers of the RuvB protein (yellow) bind to the open form of the junction. The RuvB protein, which resembles the hexameric helicases used in DNA replication (Figure 5–14), uses the energy of ATP hydrolysis to spool DNA rapidly through the Holliday junction, extending the heteroduplex region as shown. The RuvA protein coordinates this movement, threading the DNA strands to avoid tangling. (PDB codes: 1IXR, 1C7Y.) from the two Holliday junctions are thereby swapped, creating two chromosomes that have crossed over.


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Cell_Biology_Alberts. How does the cell decide which Spo11-induced double-strand breaks to resolve as crossovers? The answer is not yet known, but we know the decision is an important one. The relatively few crossovers that do form are distributed along chromosomes in such a way that a crossover in one position inhibits crossing-over in neighboring regions. Termed crossover control, this fascinating but poorly understood regulatory mechanism ensures the roughly even distribution of crossover points along chromosomes. It also ensures that each chromosome— no matter how small—undergoes at least one crossover every meiosis. For many organisms, roughly two crossovers per chromosome occur during each meiosis, one on each arm. As discussed in detail in Chapter 17, these crossovers play an important mechanical role in the proper segregation of chromosomes during meiosis.


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Cell_Biology_Alberts. Whether a meiotic recombination event is resolved as a crossover or a non-crossover, the recombination machinery leaves behind a heteroduplex region where a strand with the DNA sequence of the paternal homolog is base-paired with a strand from the maternal homolog (Figure 5–58). These heteroduplex regions can tolerate a small percentage of mismatched base pairs, and because of branch migration, they often extend for thousands of nucleotide pairs. The many non-crossover events that occur in meiosis thereby produce scattered sites in the germ cells where short DNA sequences from one homolog have been pasted into the other homolog. Heteroduplex regions mark sites of potential gene conversion—where the four haploid chromosomes produced by meiosis contain three copies of a DNA sequence from one homolog and only one copy of this sequence from the other homolog (see Figure 5–53), as explained next.


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Cell_Biology_Alberts. Figure 5–58 Heteroduplexes formed during meiosis. Heteroduplex DNA is present at sites of recombination that are resolved either as crossovers or non-crossovers. Because the DNA sequences of maternal and paternal chromosomes differ at many positions along their lengths, heteroduplexes often contain a small number of base-pair mismatches.


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Cell_Biology_Alberts. Figure 5–59 Gene conversion caused by mismatch correction. In this process, heteroduplex DNA is formed at the sites of homologous recombination between maternal and paternal chromosomes. If the maternal and paternal DNA sequences are slightly different, the heteroduplex region will include some mismatched base pairs, which may then be corrected by the DNA mismatch repair machinery (see Figure 5–19). Such repair can “erase” nucleotide sequences on either the paternal or the maternal strand. The consequence of this mismatch repair is gene conversion, detected as a deviation from the segregation of equal copies of maternal and paternal alleles that normally occurs in meiosis.


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Cell_Biology_Alberts. In sexually reproducing organisms, it is a fundamental law of genetics that—aside from mitochondrial DNA, which is inherited only through the mother—each parent makes an equal genetic contribution to an offspring. One complete set of nuclear genes is inherited from the father and one complete set is inherited from the mother. Underlying this law is the accurate parceling out of chromosomes to the germ cells (eggs and sperm) that takes place during meiosis. Thus, when a diploid cell in a parent undergoes meiosis to produce four haploid germ cells, exactly half of the genes distributed among these four cells should be maternal (genes inherited from the mother of this parent) and the other half paternal (genes inherited from the father of this parent). In some organisms (fungi, for example), it is possible to recover and analyze all four of the haploid gametes produced from a single cell by meiosis. Studies in such organisms have revealed rare cases in which the parceling out of genes


Cell_Biology_Alberts

Cell_Biology_Alberts. it is possible to recover and analyze all four of the haploid gametes produced from a single cell by meiosis. Studies in such organisms have revealed rare cases in which the parceling out of genes violates the standard genetic rules. Occasionally, for example, meiosis yields three copies of the maternal version of a gene and only one copy of the paternal allele. Alternative versions of the same gene are called alleles, and it is the divergence from their expected distribution during meiosis that is known as gene conversion. Genetic studies show that only small sections of DNA typically undergo gene conversion, and in many cases only a part of a gene is changed.


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Cell_Biology_Alberts. Several pathways in the cell can lead to gene conversion, but one of the most important arises from a particular consequence of recombination during meiosis. We have seen that both crossovers and non-crossovers produce heteroduplex regions of DNA. If the two strands that make up a heteroduplex region do not have identical nucleotide sequences, mismatched base pairs are formed, and these are often repaired by the cell’s mismatch repair system (see Figure 5–19). However, the mismatch repair system cannot distinguish between the paternal and maternal strands and will randomly choose the strand to be used as a template. As a consequence, one allele will be lost and the other duplicated (Figure 5–59), resulting in net “conversion” of one allele to the other. Thus, gene conversion, originally regarded as a mysterious deviation from the rules of genetics, can be seen as a straightforward consequence of the mechanisms of homologous recombination.


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Cell_Biology_Alberts. Homologous recombination describes a flexible set of reactions resulting in the exchange of DNA sequences between a pair of identical or nearly identical duplex DNA molecules. In all cells, this process is essential for the error-free repair of chromosome damage, particularly double-strand breaks and broken or stalled replication forks. Homologous recombination is also responsible for the crossing-over of chromosomes that occurs during meiosis. Homologous recombination takes place through a variety of pathways, but they have in common a strand-exchange step whereby a single strand from one DNA duplex invades a second duplex and base-pairs with one strand while displacing the other. This reaction, catalyzed by the RecA/Rad51 family of proteins, can only occur if the invading strand can form a short stretch of consecutive nucleotide pairs with one of the strands of the duplex. This requirement ensures that homologous recombination occurs only between identical or very similar DNA


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Cell_Biology_Alberts. form a short stretch of consecutive nucleotide pairs with one of the strands of the duplex. This requirement ensures that homologous recombination occurs only between identical or very similar DNA sequences.


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Cell_Biology_Alberts. DNA SYNTHESIS FILLS GAP, CREATING AN EXTRA COPY OF THE RED ALLELE OF When used as a repair mechanism, homologous recombination occurs between a damaged DNA molecule and its recently duplicated sister molecule, with the undamaged duplex acting as a template to repair the damaged copy flawlessly. In meiosis, homologous recombination is initiated by deliberate, carefully regulated double-strand breaks and occurs preferentially between the homologous chromosomes rather than the newly replicated sister chromatids. The outcome can be either two chromosomes that have crossed over (that is, chromosomes in which the DNA on either side of the site of DNA pairing originates from two different homo-logs) or two non-crossover chromosomes. In the latter case, the two chromosomes that result are identical to the original two homologs, except for relatively minor DNA sequence changes at the site of recombination.


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Cell_Biology_Alberts. We have seen that homologous recombination can result in the exchange of DNA sequences between chromosomes. However, the order of genes on the interacting chromosomes typically remains the same following homologous recombination, inasmuch as the recombining sequences must be very similar for the process to occur. In this section, we describe two very different types of recombination—transposition (also called transpositional recombination) and conservative site-specific recombination—that do not require substantial regions of DNA homology. These two types of recombination reactions can alter gene order along a chromosome and can cause unusual types of mutations that introduce whole blocks of DNA sequence into the genome.


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Cell_Biology_Alberts. Transposition and conservative site-specific recombination are largely dedicated to moving a wide variety of specialized segments of DNA—collectively termed mobile genetic elements—from one position in a genome to another. We will see that mobile genetic elements can range in size from a few hundred to tens of thousands of nucleotide pairs, and each typically carries a unique set of genes. Often, one of these genes encodes a specialized enzyme that catalyzes the movement of only that element, thereby making this type of recombination possible.


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Cell_Biology_Alberts. Virtually all cells contain mobile genetic elements (known informally as “jumping genes”). As explained in Chapter 4, over evolutionary time scales, they have had a profound effect on the shaping of modern genomes. For example, nearly half of the human genome can be traced to these elements (see Figure 4–62). Over time, random mutation has altered their nucleotide sequences, and, as a result, only a few of the many copies of these elements in our DNA are still active and capable of movement. The remainder are molecular fossils whose existence provides striking clues to our evolutionary history.


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Cell_Biology_Alberts. Mobile genetic elements are often considered to be molecular parasites (they are also termed “selfish DNA”) that persist because cells cannot get rid of them; they certainly have come close to overrunning our own genome. However, mobile DNA elements can provide benefits to the cell. For example, the genes they carry are sometimes advantageous, as in the case of antibiotic resistance in bacterial cells, discussed below. The movement of mobile genetic elements also produces many of the genetic variants upon which evolution depends, because, in addition to moving themselves, mobile genetic elements occasionally rearrange neighboring sequences of the host genome. Thus, spontaneous mutations observed in Drosophila, humans, and other organisms are often due to the movement of mobile genetic elements. While many of these mutations will be deleterious to the organism, some will be advantageous and may spread throughout the population. It is almost certain that much of the variety of life we


Cell_Biology_Alberts

Cell_Biology_Alberts. While many of these mutations will be deleterious to the organism, some will be advantageous and may spread throughout the population. It is almost certain that much of the variety of life we see around us originally arose from the movement of mobile genetic elements.


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Cell_Biology_Alberts. In this section, we introduce mobile genetic elements and describe the mechanisms that enable them to move around a genome. We shall see that some of these elements move through transposition mechanisms and others through conservative site-specific recombination. We begin with transposition, as there are many more known examples of this type of movement. Through Transposition, Mobile Genetic Elements Can Insert Into Any DNA Sequence


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Cell_Biology_Alberts. Through Transposition, Mobile Genetic Elements Can Insert Into Any DNA Sequence Mobile elements that move by way of transposition are called transposons, or transposable elements. In transposition, a specific enzyme, usually encoded by the transposon itself and typically called a transposase, acts on specific DNA sequences at each end of the transposon, causing it to insert into a new target DNA site. Most transposons are only modestly selective in choosing their target site, and they can therefore insert themselves into many different locations in a genome. In particular, there is no general requirement for sequence similarity between the ends of the element and the target sequence. Most transposons move only rarely. In bacteria, where it is possible to measure the frequency accurately, transposons typically move once every 105 cell divisions. More frequent movement would probably destroy the host cell’s genome.


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Cell_Biology_Alberts. On the basis of their structure and transposition mechanism, transposons can be grouped into three large classes: DNA-only transposons, retroviral-like retrotransposons, and nonretroviral retrotransposons. The differences among them are briefly outlined in Table 5–4, and each class will be discussed in turn.


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Cell_Biology_Alberts. DNA-only transposons, so named because they exist only as DNA during their movement, predominate in bacteria, and they are largely responsible for the spread of antibiotic resistance in bacterial strains. When antibiotics like penicillin and streptomycin first became widely available in the 1950s, most bacteria that caused human disease were susceptible to them. Now, the situation is different— antibiotics such as penicillin (and its modern derivatives) are no longer effective against many modern bacterial strains, including those causing gonorrhea and bacterial pneumonia. The spread of antibiotic resistance is due largely to genes that encode antibiotic-inactivating enzymes that are carried on transposons (Figure 5–60). Although these mobile elements can transpose only within cells that already carry them, they can be moved from one cell to another through other mechanisms known collectively as horizontal gene transfer (see Figure 1–19). Once introduced into a new cell, a transposon


Cell_Biology_Alberts

Cell_Biology_Alberts. already carry them, they can be moved from one cell to another through other mechanisms known collectively as horizontal gene transfer (see Figure 1–19). Once introduced into a new cell, a transposon can insert itself into the genome and be faithfully passed on to all progeny cells through the normal processes of DNA replication and cell division.


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Cell_Biology_Alberts. DNA-only transposons can relocate from a donor site to a target site by cutand-paste transposition (Figure 5–61). Here, the transposon is literally excised from one spot on a genome and inserted into another. This reaction produces a short duplication of the target DNA sequence at the insertion site; these direct repeat sequences that flank the transposon serve as convenient records of prior transposition events. Such “signatures” often provide valuable clues in identifying transposons in genome sequences.


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Cell_Biology_Alberts. When a cut-and-paste DNA-only transposon is excised from its original location, it leaves behind a “hole” in the chromosome. This lesion can be perfectly healed by recombinational double-strand break repair (see Figure 5–48), provided that the chromosome has just been replicated and an identical copy of the damaged host sequence is available. Alternatively, a nonhomologous end-joining reaction can reseal the break; in this case, the DNA sequence that originally flanked the transposon is altered, producing a mutation at the chromosomal site from which the transposon was excised (see Figure 5–45). Remarkably, the same mechanism used to excise cut-and-paste transposons from DNA has been found to operate in developing immune systems of short direct repeats of target DNA sequences in chromosome B


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Cell_Biology_Alberts. Figure 5–60 Three of the many DNA-only transposons found in bacteria. Each of these mobile DNA elements contains a gene that encodes a transposase, an enzyme that carries out the DNA breakage and joining reactions needed for the element to move. Each transposon also carries short DNA sequences (indicated in red) that are recognized only by the transposase encoded by that element and are necessary for movement of the element. In addition, two of the three mobile elements shown carry genes that encode enzymes that inactivate the antibiotics ampicillin (AmpR)—a penicillin derivative—and tetracycline (TetR). The transposable element Tn10, shown in the bottom diagram, is thought to have evolved from the chance landing of two much shorter mobile elements on either side of a tetracycline-resistance gene.


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Cell_Biology_Alberts. Figure 5–61 Cut-and-paste transposition. DNA-only transposons can be recognized in chromosomes by the “inverted repeat DNA sequences” (red) present at their ends. These sequences, which can be as short as 20 nucleotides, are all that is necessary for the DNA between them to be transposed by the particular transposase enzyme associated with the element. The cut-and-paste movement of a DNA-only transposable element from one chromosomal site to another begins when the transposase brings the two inverted DNA sequences together, forming a DNA loop. Insertion into the target chromosome, also catalyzed by the transposase, occurs at a random site through the creation of staggered breaks in the target chromosome (purple arrowheads). Following the transposition reaction, the single-strand gaps created by the staggered breaks are repaired by DNA polymerase and ligase (black). As a result, the insertion site is marked by a short direct repeat of the target DNA sequence, as shown. Although the


Cell_Biology_Alberts

Cell_Biology_Alberts. by the staggered breaks are repaired by DNA polymerase and ligase (black). As a result, the insertion site is marked by a short direct repeat of the target DNA sequence, as shown. Although the break in the donor chromosome (green) is repaired, this process often alters the DNA sequence, causing a mutation at the original site of the excised transposable element (not shown).


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Cell_Biology_Alberts. vertebrates, catalyzing the DNA rearrangements that produce antibody and T cell receptor diversity. Known as V(D)J recombination, this process will be discussed in Chapter 24. Found only in vertebrates, V(D)J recombination is a relatively recent evolutionary novelty, but it is believed to be derived from the much more ancient cut-and-paste transposons. Some Viruses Use a Transposition Mechanism to Move Themselves Into Host-Cell Chromosomes


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Cell_Biology_Alberts. Some Viruses Use a Transposition Mechanism to Move Themselves Into Host-Cell Chromosomes Certain viruses are considered mobile genetic elements because they use transposition mechanisms to integrate their genomes into that of their host cell. However, unlike transposons, these viruses encode proteins that package their genetic information into virus particles that can infect other cells. Many of the viruses that insert themselves into a host chromosome do so by employing one of the first two mechanisms listed in Table 5–4; namely, by behaving like DNA-only transposons or like retroviral-like retrotransposons. Indeed, much of our knowledge of these mechanisms has come from studies of particular viruses that employ them.


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Cell_Biology_Alberts. Transposition has a key role in the life cycle of many viruses. Most notable are the retroviruses, which include the human AIDS virus, HIV. Outside the cell, a retrovirus exists as a single-strand RNA genome packed into a protein shell or capsid along with a virus-encoded reverse transcriptase enzyme. During the infection process, the viral RNA enters a cell and is converted to a double-strand DNA molecule by the action of this crucial enzyme, which is able to polymerize DNA on either an RNA or a DNA template (Figure 5–62). The term retrovirus refers to the virus’s ability to reverse the usual flow of genetic information, which normally is from DNA to RNA (see Figure 1–4). Once the reverse transcriptase has produced a double-strand DNA molecule, specific sequences near its two ends are recognized by a virus-encoded


Cell_Biology_Alberts

Cell_Biology_Alberts. Once the reverse transcriptase has produced a double-strand DNA molecule, specific sequences near its two ends are recognized by a virus-encoded Figure 5–62 The life cycle of a retrovirus. The retrovirus genome consists of an RNA molecule (blue) that is typically between 7000 and 12,000 nucleotides in length. It is packaged inside a protein capsid, which is surrounded by a lipid-based envelope that contains virus-encoded envelope proteins (green). Inside an infected cell, the enzyme reverse transcriptase (red circle) first makes a DNA copy of the viral RNA molecule and then a second DNA strand, generating a double-strand DNA copy of the RNA genome. The integration of this DNA double helix into the host chromosome is then catalyzed by a virus-encoded integrase enzyme. This integration is required for the synthesis of new viral RNA molecules by the host-cell RNA polymerase, the enzyme that transcribes DNA into RNA (discussed in Chapter 6).


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Cell_Biology_Alberts. transposase called integrase. Integrase then inserts the viral DNA into the chromosome by a mechanism similar to that used by the cut-and-paste DNA-only transposons (see Figure 5–61). Retroviral-like Retrotransposons Resemble Retroviruses, but Lack a Protein Coat


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Cell_Biology_Alberts. A large family of transposons called retroviral-like retrotransposons (see Table 5–4) move themselves in and out of chromosomes by a mechanism that is similar to that used by retroviruses. These elements are present in organisms as diverse as yeasts, flies, and mammals; unlike viruses, they have no intrinsic ability to leave their resident cell but are passed along to all descendants of that cell through the normal processes of DNA replication and cell division. The first step in their transposition is the transcription of the entire transposon, producing an RNA copy of the element that is typically several thousand nucleotides long. This transcript, which is translated as a messenger RNA by the host cell, encodes a reverse transcriptase enzyme. This enzyme makes a double-strand DNA copy of the RNA molecule via an RNA–DNA hybrid intermediate, precisely mirroring the early stages of infection by a retrovirus (see Figure 5–62). Like a retrovirus, the linear, double-strand DNA molecule


Cell_Biology_Alberts

Cell_Biology_Alberts. of the RNA molecule via an RNA–DNA hybrid intermediate, precisely mirroring the early stages of infection by a retrovirus (see Figure 5–62). Like a retrovirus, the linear, double-strand DNA molecule then integrates into a site on the chromosome using an integrase enzyme that is also encoded by the element. The structure and mechanisms of these integrases closely resemble those of the transposases of DNA-only transposons.


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Cell_Biology_Alberts. A Large Fraction of the Human Genome Is Composed of Nonretroviral Retrotransposons A significant fraction of many vertebrate chromosomes is made up of repeated DNA sequences. In human chromosomes, these repeats are mostly mutated and truncated versions of nonretroviral retrotransposons, the third major type of transposon (see Table 5–4). Although most of these transposons in the human genome are immobile, a few retain the ability to move. Relatively recent movements of the L1 element (sometimes referred to as a LINE or long interspersed nuclear element) have been identified, some of which result in human disease; for example, a particular type of hemophilia results from an L1 insertion into the gene encoding the blood-clotting protein Factor VIII (see Figure 6–24). binds to L1 RNA


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Cell_Biology_Alberts. binds to L1 RNA Nonretroviral retrotransposons are found in many organisms and move via a 5˜ distinct mechanism that requires a complex of an endonuclease and a reverse transcriptase. As illustrated in Figure 5–63, the RNA and reverse transcriptase CLEAVAGE OF FIRST STRAND have a much more direct role in the recombination event than they do in the ret- roviral-like retrotransposons described above.


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Cell_Biology_Alberts. Inspection of the human genome sequence reveals that the bulk of nonretro ber of the SINE (short interspersed nuclear element) family—do not carry their viral retrotransposons—for example, the many copies of the Alu element, a mem-target DNA own endonuclease or reverse transcriptase genes. Nonetheless, they have suc cessfully amplified themselves to become major constituents of our genome, pre- sumably by pirating enzymes encoded by other transposons. Together the LINEs and SINEs make up over 30% of the human genome (see Figure 4–62); there are 500,000 copies of the former and over a million of the latter. Figure 5–63 Transposition by a nonretroviral retrotransposon.


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Cell_Biology_Alberts. Figure 5–63 Transposition by a nonretroviral retrotransposon. Transposition of the L1 element (red) begins when an endonuclease attached to the L1 reverse transcriptase (green) and the L1 RNA (blue) nick the target DNA at the point at which insertion will occur. This cleavage releases a 3ʹ-OH DNA end in the target DNA, which is then used as a primer for the reverse transcription step shown. This generates a single-strand DNA copy of the 3˜ element that is directly linked to the target DNA. In subsequent reactions, further processing of the single-strand DNA copy results in the generation of a new double-strand DNA copy of the L1 element that is inserted at the site of the initial nick.


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Cell_Biology_Alberts. We have described several types of transposable elements: (1) DNA-only transposons, the movement of which is based on DNA breaking and joining reactions; (2) retroviral-like retrotransposons, which also move via DNA breakage and joining, but where RNA has a key role as a template to generate the DNA recombination substrate; and (3) nonretroviral retrotransposons, in which an RNA copy of the element is central to the incorporation of the element into the target DNA, acting as a direct template for a DNA target-primed reverse transcription event.


Cell_Biology_Alberts

Cell_Biology_Alberts. Intriguingly, different types of transposons predominate in different organisms. For example, the vast majority of bacterial transposons are DNA-only types, with a few related to the nonretroviral retrotransposons also present. In yeasts, the main mobile elements are retroviral-like retrotransposons. In Drosophila, DNA-based, retroviral, and nonretroviral transposons are all found. Finally, the human genome contains all three types of transposon, but as discussed below, their evolutionary histories are strikingly different. Genome Sequences Reveal the Approximate Times at Which Transposable Elements Have Moved


Cell_Biology_Alberts

Cell_Biology_Alberts. The nucleotide sequence of the human genome provides a rich fossil record of the activity of transposons over evolutionary time spans. By carefully comparing the nucleotide sequences of the approximately 3 million transposable element remnants in the human genome, it has been possible to broadly reconstruct the movements of transposons in our ancestors’ genomes over the past several hundred million years. For example, the DNA-only transposons appear to have been very active well before the divergence of humans and Old World monkeys (25–35 million years ago), but because they gradually accumulated inactivating mutations, they have been dormant in the human lineage since that time. Likewise, although our genome is littered with relics of retroviral-like retrotransposons, none appear to be active today. Only a single family of retroviral-like retrotransposons is believed to have transposed in the human genome since the divergence of human and chimpanzee approximately 6 million years ago.


Cell_Biology_Alberts

Cell_Biology_Alberts. today. Only a single family of retroviral-like retrotransposons is believed to have transposed in the human genome since the divergence of human and chimpanzee approximately 6 million years ago. The nonretroviral retrotransposons are also ancient, but in contrast to other types, some are still moving in our genome, as mentioned previously. For example, it is estimated that de novo movement of an Alu element is seen once in every 100–200 human births. The movement of nonretroviral retrotransposons is responsible for a small but significant fraction of new human mutations—perhaps two mutations out of every thousand.


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Cell_Biology_Alberts. The situation in mice is significantly different. Although the mouse and human genomes contain roughly the same density of the three types of transposons, both types of retrotransposons are still actively transposing in the mouse genome, being responsible for approximately 10% of new mutations. Although we are only beginning to understand how the movements of transposons have shaped the genomes of present-day mammals, it has been proposed that bursts in transposition activity could have been responsible for critical speciation events during the radiation of the mammalian lineages from a common ancestor, a process that began approximately 170 million years ago. At present, we can only wonder how many of our uniquely human qualities arose from the past activity of the many mobile genetic elements whose remnants are found today scattered throughout our chromosomes.


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Cell_Biology_Alberts. A different kind of recombination mechanism, known as conservative site-specific recombination, rearranges other types of mobile DNA elements. In this pathway, breakage and joining occur at two special sites, one on each participating DNA Figure 5–64 Two types of DNA rearrangement produced by conservative site-specific recombination. The only difference between the reactions in (A) and (B) is the relative orientation of the two short DNA sites (indicated by arrows) at which a site-specific recombination event occurs. Through an integration reaction, a circular DNA molecule can become incorporated into a second DNA molecule; by the reverse reaction (excision), it can exit to re-form the original DNA circle. Many bacterial viruses move in and out of their host chromosomes in this way.


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Cell_Biology_Alberts. Conservative site-specific recombination can also invert a specific segment of DNA in a chromosome. A well-studied example of DNA inversion through site-specific recombination occurs in the bacterium Salmonella typhimurium, an organism that is a major cause of food poisoning in humans; as described in the following section, the inversion of a DNA segment changes the type of flagellum that is produced by the bacterium. molecule. Depending on the positions and relative orientations of the two recombination sites, DNA integration, DNA excision, or DNA inversion can occur (Figure 5–64). Conservative site-specific recombination is carried out by specialized enzymes that break and rejoin two DNA double helices at specific sequences on each DNA molecule. The same enzyme system that joins two DNA molecules can often take them apart again, precisely restoring the sequence of the two original DNA molecules (see Figure 5–64A).


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Cell_Biology_Alberts. Conservative site-specific recombination is often used by DNA viruses to move their genomes in and out of the genomes of their host cells. When integrated into its host genome, the viral DNA is replicated along with the host DNA and is faithfully passed on to all descendent cells. If the host cell suffers damage (for example, by UV irradiation), the virus can reverse the site-specific recombination reaction, excise its genome, and package it into a virus particle. In this way, many viruses can replicate themselves passively as a component of the host genome, but can also “leave the sinking ship” by excising their genomes and packaging them in a protective coat until a new, healthy host cell is encountered.


Cell_Biology_Alberts

Cell_Biology_Alberts. Several features distinguish conservative site-specific recombination from transposition. First, conservative site-specific recombination requires specialized DNA sequences on both the donor and recipient DNA (hence the term site-specific). These sequences contain recognition sites for the particular recombinase that will catalyze the rearrangement. In contrast, transposition requires only that the transposon have a specialized sequence; for most transposons, the recipient DNA can be of any sequence. Second, the reaction mechanisms are fundamentally different. The recombinases that catalyze conservative site-specific recombination resemble topoisomerases in the sense that they form transient high-energy covalent bonds with the DNA and use this energy to complete the DNA rearrangements (see Figure 5–21). Thus, all the phosphate bonds that are broken during a recombination event are restored upon its completion (hence the term conservative). Transposition, in contrast, does not proceed


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Cell_Biology_Alberts. Figure 5–21). Thus, all the phosphate bonds that are broken during a recombination event are restored upon its completion (hence the term conservative). Transposition, in contrast, does not proceed through a covalently joined protein–DNA intermediate, and this process leaves gaps in the DNA that must be repaired by DNA polymerases.


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Cell_Biology_Alberts. Conservative Site-Specific Recombination Can Be Used to Turn Genes On or Off


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Cell_Biology_Alberts. Many bacteria use conservative site-specific recombination to control the expression of particular genes. A well-studied example occurs in Salmonella bacteria and is known as phase variation. The switch in gene expression results from the occasional inversion of a specific 1000-nucleotide-pair piece of DNA, brought about by a conservative site-specific recombinase encoded in the Salmonella genome. This change alters the expression of the cell-surface protein flagellin, for which the bacterium has two different genes (Figure 5–65). The DNA inversion changes the orientation of a promoter (a DNA sequence that directs transcription of a gene) that is located within the inverted DNA segment. With the promoter in one orientation, the bacteria synthesize one type of flagellin; with the promoter in the other orientation, they synthesize the other type. The recombination reaction is reversible, allowing bacterial populations to switch back and forth between the two types of flagellin.


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Cell_Biology_Alberts. in the other orientation, they synthesize the other type. The recombination reaction is reversible, allowing bacterial populations to switch back and forth between the two types of flagellin. Inversions occur only rarely, and because such changes in the genome will be copied faithfully during all subsequent replication cycles, entire clones of bacteria will have one type of flagellin or the other.


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Cell_Biology_Alberts. Phase variation helps protect the bacterial population against the immune response of its vertebrate host. If the host makes antibodies against one type of flagellin, a few bacteria whose flagellin has been altered by gene inversion will still be able to survive and multiply.


Cell_Biology_Alberts

Cell_Biology_Alberts. Like many of the mechanisms used by cells and viruses, site-specific recombination has been put to work by scientists to study a wide variety of problems. To decipher the roles of specific genes and proteins in complex multicellular organisms, genetic engineering techniques are used to produce worms, flies, and mice carrying a gene encoding a site-specific recombination enzyme plus a carefully designed target DNA with the DNA sites that this enzyme recognizes. At an appropriate time, the gene encoding the enzyme can be activated to rearrange the target DNA sequence. Such a rearrangement is widely used to delete a specific gene in a particular tissue of a multicellular organism (Figure 5–66). It is particularly useful when the gene of interest plays a key role in the early development of many tissues, and a complete deletion of the gene from the germ line would cause death Figure 5–65 Switching gene expression by DNA inversion in bacteria.


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Cell_Biology_Alberts. Figure 5–65 Switching gene expression by DNA inversion in bacteria. Alternating transcription of two flagellin genes in a Salmonella bacterium is caused by a conservative site-specific recombination event that inverts a small DNA segment containing a promoter. In one orientation, the promoter activates transcription of the H2 flagellin gene as well as that of a repressor protein that blocks the expression of the H1 flagellin gene. Promoters and repressors are described in detail in Chapter 7; here we note simply that a promoter is needed to express a gene into protein and that a repressor blocks this from happening.


Cell_Biology_Alberts

Cell_Biology_Alberts. When the promoter is inverted, it no longer turns on H2 or the repressor, and the H1 gene, which is thereby released from repression, is expressed instead. The inversion reaction requires specific DNA sequences (red) and a recombinase enzyme that is encoded in the invertible DNA segment. This site-specific recombination mechanism is activated only rarely (about once in every 105 cell divisions). Therefore, the production of one or the other flagellin tends to be faithfully inherited in each clone of cells. IN SPECIFIC TISSUE (e.g., LIVER) Cre recombinase gene gene of interest gene of interest deleted from chromosome and lost as liver cells divide IN OTHER TISSUES, THE GENE OF INTEREST IS EXPRESSED NORMALLY Cre recombinase gene gene of interest mRNA(e.g., promoter active only in liver) protein of interest


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Cell_Biology_Alberts. Figure 5–66 How a conservative site-specific recombination enzyme from bacteria is used to delete specific genes from particular mouse tissues. This approach requires the insertion of two specially engineered DNA molecules into the animal’s germ line. The first contains the gene for a recombinase (in this case, the Cre recombinase from the bacteriophage P1) under the control of a tissue-specific promoter, which ensures that the recombinase is expressed only in that tissue. The second DNA molecule contains the gene of interest flanked by recognition sites (in this case, LoxP sites) for the recombinase. The mouse is engineered so that this is the only copy of this gene. Therefore, if the recombinase is expressed only in the liver, the gene of interest will be deleted there, and only there. The reaction that excises the gene is the same as that shown in Figure 5–64A. As described in Chapter 7, many tissue-specific promoters are known; moreover, many of these promoters are active only at


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Cell_Biology_Alberts. The reaction that excises the gene is the same as that shown in Figure 5–64A. As described in Chapter 7, many tissue-specific promoters are known; moreover, many of these promoters are active only at specific times in development. Thus, it is possible to study the effect of deleting specific genes at different times during the development of each tissue.


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Cell_Biology_Alberts. • How does DNA replication contend with all the other processes that occur simultaneously on chromosomes, very early in development. The same strategy can also be used to inappropriately including DNA repair and gene express any specific gene in a tissue of interest; here, the triggered deletion joins transcription? a strong transcriptional promoter to the gene of interest. With this tool one can in principle determine the influence of any protein in any desired tissue of an intact • What is the basis for the low animal. frequency of errors in DNA replication observed in all cells? Is this the best that cells can do given the speed of replication and the limits of molecular The genomes of nearly all organisms contain mobile genetic elements that can move diffusion? Was this mutation rate from one position in the genome to another by either transpositional or conserva-selected in evolution to provide genetic variation?


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Cell_Biology_Alberts. tive site-specific recombination processes. In most cases, this movement is random and happens at a very low frequency. Mobile genetic elements include transposons, which move within a single cell (and its descendants), plus those viruses whose genomes can integrate into the genomes of their host cells. ways of repairing it. Are there still There are three classes of transposons: the DNA-only transposons, the retrovi other, undiscovered ways that cells ral-like retrotransposons, and the nonretroviral retrotransposons. All but the last have for repairing DNA? have close relatives among the viruses. Although viruses and transposable elements can be viewed as parasites, many of the new arrangements of DNA sequences that • Do the many “dead” transposons their site-specific recombination events produce have played an important part in in the human genome provide any creating the genetic variation crucial for the evolution of cells and organisms. benefits to humans?


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Cell_Biology_Alberts. Which statements are true? Explain why or why not. 5–1 The different cells in your body rarely have genomes with the identical nucleotide sequence. 5–2 In E. coli, where the replication fork travels at 500 nucleotide pairs per second, the DNA ahead of the fork— in the absence of topoisomerase—would have to rotate at nearly 3000 revolutions per minute. 5–3 In a replication bubble, the same parental DNA strand serves as the template strand for leading-strand synthesis in one replication fork and as the template for lagging-strand synthesis in the other fork. 5–4 When bidirectional replication forks from adjacent origins meet, a leading strand always runs into a lagging strand. 5–5 DNA repair mechanisms all depend on the existence of two copies of the genetic information, one in each of the two homologous chromosomes. Discuss the following problems.


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Cell_Biology_Alberts. 5–5 DNA repair mechanisms all depend on the existence of two copies of the genetic information, one in each of the two homologous chromosomes. Discuss the following problems. 5–6 To determine the reproducibility of mutation frequency measurements, you do the following experiment. You inoculate each of 10 cultures with a single E. coli bacterium, allow the cultures to grow until each contains 106 cells, and then measure the number of cells in each culture that carry a mutation in your gene of interest. You were so surprised by the initial results that you repeated the experiment to confirm them. Both sets of results display the same extreme variability, as shown in Table Q5–1. Assuming that the rate of mutation is constant, why do you suppose there is so much variation in the frequencies of mutant cells in different cultures?


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Cell_Biology_Alberts. 5–7 DNA repair enzymes preferentially repair mismatched bases on the newly synthesized DNA strand, using the old DNA strand as a template. If mismatches were instead repaired without regard for which strand served as template, would mismatch repair reduce replication errors? Would such a mismatch repair system result in fewer mutations, more mutations, or the same number of mutations as there would have been without any repair at all? Explain your answers. 5–8 Discuss the following statement: “Primase is a sloppy enzyme that makes many mistakes. Eventually, the RNA primers it makes are replaced with DNA made by a polymerase with higher fidelity. This is wasteful. It would be more energy-efficient if a DNA polymerase made an accurate copy in the first place.” 5–9 If DNA polymerase requires a perfectly paired primer in order to add the next nucleotide, how is it that any mismatched nucleotides “escape” this requirement and become substrates for mismatch repair enzymes?


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Cell_Biology_Alberts. 5–10 The laboratory you joined is studying the life cycle of an animal virus that uses circular, double-strand DNA as its genome. Your project is to define the location of the origin(s) of replication and to determine whether replication proceeds in one or both directions away from an origin (unidirectional or bidirectional replication). To accomplish your goal, you broke open cells infected with the virus, isolated replicating viral genomes, cleaved them with a restriction nuclease that cuts the genome at only one site to produce a linear molecule from the circle, and examined the resulting molecules in the electron microscope. Some of the molecules you observed are illustrated schematically in Figure Q5–1. (Note that it is impossible to distinguish the orientation of one DNA molecule relative to another in the electron microscope.)


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Cell_Biology_Alberts. You must present your conclusions to the rest of the lab tomorrow. How will you answer the two questions your advisor posed for you? Is there a single, unique origin of replication or several origins? Is replication unidirectional or bidirectional? Figure Q5–1 Parental and replicating forms of an animal virus (Problem 5–10). 5–11 You are investigating DNA synthesis in tissue-culture cells, using 3H-thymidine to radioactively label the replication forks. By breaking open the cells in a way that allows some of the DNA strands to be stretched out, very long DNA strands can be isolated intact and examined. You overlay the DNA with a photographic emulsion, and expose it for 3 to 6 months, a procedure known as auto-radiography. Because the emulsion is sensitive to radioactive emissions, the 3H-labeled DNA shows up as tracks of silver grains. Because the stretching collapses replication


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Cell_Biology_Alberts. Figure Q5–2 Autoradiographic investigation of DNA replication in cultured cells (Problem 5–11). (A) Addition of 3H-labeled thymidine immediately after release from the synchronizing block. (B) Addition of 3H-labeled thymidine 30 minutes after release from the synchronizing block. bubbles, the daughter duplexes lie side by side and cannot be distinguished from each other.


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Cell_Biology_Alberts. bubbles, the daughter duplexes lie side by side and cannot be distinguished from each other. You pretreat the cells to synchronize them at the beginning of S phase. In the first experiment, you release the synchronizing block and add 3H-thymidine immediately. After 30 minutes, you wash the cells and change the medium so that the total concentration of thymidine is the same as it was, but only one-third of it is radioactive. After an additional 15 minutes, you prepare DNA for autoradiography. The results of this experiment are shown in Figure Q5–2A. In the second experiment, you release the synchronizing block and then wait 30 minutes before adding 3H-thymidine. After 30 minutes in the presence of 3H-thymidine, you once again change the medium to reduce the concentration of radioactive thymidine and incubate the cells for an additional 15 minutes. The results of the second experiment are shown in Figure Q5–2B.


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Cell_Biology_Alberts. A. Explain why, in both experiments, some regions of the tracks are dense with silver grains (dark), whereas others are less dense (light). b. In the first experiment, each track has a central dark section with light sections at each end. In the second experiment, the dark section of each track has a light section at only one end. Explain the reason for this difference. C. Estimate the rate of fork movement (μm/min) in these experiments. Do the estimates from the two experiments agree? Can you use this information to gauge how long it would take to replicate the entire genome?


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Cell_Biology_Alberts. 5–12 If you compare the frequency of the sixteen possible dinucleotide sequences in the E. coli and human genomes, there are no striking differences except for one dinucleotide, 5ʹ-CG-3ʹ. The frequency of CG dinucleotides in the human genome is significantly lower than in E. coli and significantly lower than expected by chance. Why do you suppose that CG dinucleotides are underrepresented in the human genome? 5–13 With age, somatic cells are thought to accumulate genomic “scars” as a result of the inaccurate repair of double-strand breaks by nonhomologous end joining (NHEJ).


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Cell_Biology_Alberts. Estimates based on the frequency of breaks in primary human fibroblasts suggest that by age 70, each human somatic cell may carry some 2000 NHEJ-induced mutations due to inaccurate repair. If these mutations were distributed randomly around the genome, how many pro-tein-coding genes would you expect to be affected? Would you expect cell function to be compromised? Why or why not? (Assume that 2% of the genome—1.5% protein-coding and 0.5% regulatory—is crucial information.) 5–14 Draw the structure of the double Holliday junction that would result from strand invasion by both ends of the broken duplex into the intact homologous duplex shown in Figure Q5–3. Label the left end of each strand in the Holliday junction 5ʹor 3ʹ so that the relationship to the parental and recombinant duplexes is clear. Indicate how DNA synthesis would be used to fill in any single-strand gaps in your double Holliday junction. (Problem 5–14).


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Cell_Biology_Alberts. (Problem 5–14). 5–15 In addition to correcting DNA mismatches, the mismatch repair system functions to prevent homologous recombination from taking place between similar but not identical sequences. Why would recombination between similar, but nonidentical sequences pose a problem for human cells? 5–16 Cre recombinase is a site-specific enzyme that catalyzes recombination between two LoxP DNA sites. Cre recombinase pairs two LoxP sites in the same orientation, breaks both duplexes at the same point in each LoxP site, and joins the ends with new partners so that each LoxP site is regenerated, as shown schematically in Figure Q5–4A. Based on this mechanism, predict the arrangement of sequences that will be generated by Cre-mediated site-specific recombination for each of the two DNAs shown in Figure Q5–4B.


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Cell_Biology_Alberts. Figure Q5–4 Cre recombinase-mediated site-specific recombination (Problem 5–16). (A) Schematic representation of Cre/LoxP site-specific recombination. The LoxP sequences in the DNA are represented by triangles that are colored so that the site-specific recombination event can be followed more readily. In reality their DNA sequences are identical. (B) DNA substrates containing two arrangements of LoxP sites. Brown TA (2007) Genomes 3. New York: Garland Science. Friedberg EC, Walker GC, Siede W et al. (2005) DNA Repair and Mutagenesis. Washington, DC: ASM Press. Haber JE (2013) Genome Stability: DNA Repair and Recombination. New York: Garland Science. Hartwell L, Hood L, Goldberg ML et al. (2010) Genetics: from Genes to Genomes. Boston: McGraw Hill. Stent GS (1971) Molecular Genetics: An Introductory Narrative. San Francisco: WH Freeman. Watson J, Baker T, Bell S et al. (2013) Molecular Biology of the Gene, 7th ed. Menlo Park, CA: Benjamin Cummings.


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Cell_Biology_Alberts. Watson J, Baker T, Bell S et al. (2013) Molecular Biology of the Gene, 7th ed. Menlo Park, CA: Benjamin Cummings. The Maintenance of DNA Sequences Conrad DF, Keebler J, DePristo M et al. (2011) Variation in genome-wide mutation rates within and between human families. Nat. Genet. 43, 712–714. Catarina D & Eichler EE (2013) Properties and rates of germline mutations in humans. Trends Genet. 29, 575–584. Cooper GM, Brudno M, Stone ES et al. (2004) Characterization of evolutionary rates and constraints in three mammalian genomes. Genome Res. 14, 539–548. Hedges SB (2002) The origin and evolution of model organisms. Nat. Rev. Genet. 3, 838–849. King MC & Wilson AC (1965) Evolution at two levels in humans and chimpanzees. Science 188, 107–116. Alberts B (1998) The cell as a collection of protein machines: preparing the next generation of molecular biologists. Cell 92, 291–294.


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Cell_Biology_Alberts. Alberts B (1998) The cell as a collection of protein machines: preparing the next generation of molecular biologists. Cell 92, 291–294. Kelch BA, Makino DL, O’Donnell M et al. (2011) How a DNA polymerase clamp loader opens a sliding clamp. Science 334, 1675–1680. Kornberg A (1960) Biological synthesis of DNA. Science 131, 1503– 1508. Li JJ & Kelly TJ (1984) SV40 DNA replication in vitro. Proc. Natl. Acad. Sci. USA 81, 6973–6977. Meselson M & Stahl FW (1958) The replication of DNA in E. coli. Proc. Natl. Acad. Sci. USA 44, 671–682. Modrich P & Lahue R (1996) Mismatch repair in replication fidelity, genetic recombination, and cancer biology. Annu. Rev. Biochem. 65, 101–133. O’Donnell M, Langston L & Stillman B (2013) Principals and concepts of DNA replication in Bacteria, Archaea, and Eukarya. Cold Spring Harb. Lab. Perspect. Biol. 195, 1231–1240.


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Cell_Biology_Alberts. O’Donnell M, Langston L & Stillman B (2013) Principals and concepts of DNA replication in Bacteria, Archaea, and Eukarya. Cold Spring Harb. Lab. Perspect. Biol. 195, 1231–1240. Okazaki R, Okazaki T, Sakabe K et al. (1968) Mechanism of DNA chain growth. I. Possible discontinuity and unusual secondary structure of newly synthesized chains. Proc. Natl. Acad. Sci. USA 59, 598–605. Raghuraman MK, Winzeler EA, Collingwood D et al. (2001) Replication dynamics of the yeast genome. Science 294, 115–121. Rao PN & Johnson RT (1970) Mammalian cell fusion: studies on the regulation of DNA synthesis and mitosis. Nature 225, 159. Vos SM, Tretter EM, Schmidt BH et al. (2011) All tangled up: how cells direct, manage and exploit topoisomerase function. Nat. Rev. Mol. Cell Biol. 12, 827–841. The Initiation and Completion of DNA Replication in Chromosomes Chan SR & Blackburn EH (2004) Telomeres and telomerase. Philos. Trans. R. Soc. Lond. B Bio. Sci. 359, 109–121.


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Cell_Biology_Alberts. The Initiation and Completion of DNA Replication in Chromosomes Chan SR & Blackburn EH (2004) Telomeres and telomerase. Philos. Trans. R. Soc. Lond. B Bio. Sci. 359, 109–121. Gilbert DM (2010) Evaluating genome-scale approaches to eukaryotic DNA replication. Nat. Rev. Genet. 11, 673–684. deLang T (2009) How telomeres solve the end-protection problem. Science 326, 948–952. Mechali M (2010) Eukaryotic DNA replication origins: many choices for appropriate answers. Nat. Rev. Mol. Cell Biol. 11, 728–738. Nandakumar J & Cech T (2013) Finding the end: recruitment of telomerase to telomeres. Nat. Rev. Mol. Cell Biol. 14, 69–82. Goodman MF & Woodgate, R (2013) Translesion DNA polymerases. Cold Spring Harb. Perspect. Biol. 5, a010363. Hanawalt PC & Spivak G (2008) Transcription-coupled DNA repair: two decades of progress and surprises. Nat. Rev. Mol. Cell Biol. 9, 958–970. Lindahl T (1993) Instability and decay of the primary structure of DNA. Nature 362, 709–715.


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Cell_Biology_Alberts. Lindahl T (1993) Instability and decay of the primary structure of DNA. Nature 362, 709–715. Malkova A & Haber JE (2012) Mutations arising during repair of chromosome breaks. Annu. Rev. Genet. 46, 455–473. Prakash S, Johnson RE & Prakash L (2005) Eukaryotic translesion synthesis DNA polymerases: specificity of structure and function. Annu. Rev. Biochem. 74, 317–353. Reardon JT & Sancar A (2005) Nucleotide excision repair. Prog. Nucleic Acid Res. Mol. Biol. 79, 183–235. Chen Z, Yang H & Pavletich NP (2008) Mechanism of homologous recombination from the RecA-ssDNA/dsDNA structures. Nature 453, 489–494. Cox MM (2001) Historical overview: searching for replication help in all of the rec places. Proc. Natl. Acad. Sci. USA 98, 8173–8180. Heyer WD, Ehmsen KT & Liu J (2010) Regulation of homologous recombination in eukaryotes. Annu. Rev. Genet. 44, 113–139. Holliday R (1990) The history of the DNA heteroduplex. BioEssays 12, 133–142.


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Cell_Biology_Alberts. Heyer WD, Ehmsen KT & Liu J (2010) Regulation of homologous recombination in eukaryotes. Annu. Rev. Genet. 44, 113–139. Holliday R (1990) The history of the DNA heteroduplex. BioEssays 12, 133–142. Hunter N (2006) Meiotic recombination. In Topics in Current Genetics, Molecular Genetics of Recombination, Aguilera A & Rothstein R (eds), pp. 381–422. Springer-Verlag: Heidelberg. de Massy B (2013) Initiation of meiotic recombination: how and where? Conservation and specificities among eukaryotes. Annu. Rev. Genet. 47, 563–599. Michel B, Gromponee G, Florès MJ & Bidnenko V (2004) Multiple pathways process stalled replication forks. Proc. Natl. Acad. Sci. USA 101, 12783–12788. Moynahan ME & Jasin M (2010) Mitotic homologous recombination maintains genomic stability and suppresses tumorigenesis. Nat. Rev. Mol. Cell Biol. 11, 196–207. Szostak JW, Orr-Weaver TK, Rothstein RJ et al. (1983) The double-strand break repair model for recombination. Cell 33, 25–35.


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Cell_Biology_Alberts. Szostak JW, Orr-Weaver TK, Rothstein RJ et al. (1983) The double-strand break repair model for recombination. Cell 33, 25–35. West SC (2003) Molecular views of recombination proteins and their control. Nat. Rev. Mol. Cell Biol. 4(6), 435–445. Yeeles JY, Poli J, Marians KJ et al. (2013) Rescuing stalled or damaged replication forks. Cold Spring Harb. Perspect. Biol. 5, a012815. Zickler D & Kleckner N (1999) Meiotic chromosomes: integrating structure and function. Annu. Rev. Genet. 33, 603–754. Comfort NC (2001) From controlling elements to transposons: Barbara McClintock and the Nobel Prize. Trends Biochem. Sci. 26, 454–457. Grindley ND, Whiteson KL & Rice PA (2006) Mechanisms of site-specific recombination. Annu. Rev. Biochem. 75, 567–605. Huang, CR, Burns KH & Boeke JD (2012) Active transposition in genomes. Annu. Rev. Genet. 46, 651–675. Varmus H (1988) Retroviruses. Science 240, 1427–1435. How Cells Read the Genome: From DNA to Protein


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Cell_Biology_Alberts. Varmus H (1988) Retroviruses. Science 240, 1427–1435. How Cells Read the Genome: From DNA to Protein Since the structure of DNA was discovered in the early 1950s, progress in cell and molecular biology has been astounding. We now know the complete genome sequences for thousands of different organisms, revealing fascinating details of their biochemistry as well as important clues as to how these organisms evolved. Complete genome sequences have also been obtained for thousands of individual humans, as well as for a few of our now-extinct relatives, such as the Neanderthals. Knowing the maximum amount of information that is required to produce a complex organism like ourselves puts constraints on the biochemical and structural features of cells and makes it clear that biology is not infinitely complex.


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Cell_Biology_Alberts. As discussed in Chapter 1, the DNA in genomes does not direct protein synthesis itself, but instead uses RNA as an intermediary. When the cell needs a particular protein, the nucleotide sequence of the appropriate portion of the immensely long DNA molecule in a chromosome is first copied into RNA (a process called transcription). It is these RNA copies of segments of the DNA that are used directly as templates to direct the synthesis of the protein (a process called translation). The flow of genetic information in cells is therefore from DNA to RNA to protein (Figure 6–1). All cells, from bacteria to humans, express their genetic information in this way—a principle so fundamental that it is termed the central dogma of molecular biology. Despite the universality of the central dogma of molecular biology, there are important variations between organisms in the way in which information flows from DNA to protein. Principal among these is that RNA transcripts in eukaryotic cells are


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Cell_Biology_Alberts. of molecular biology, there are important variations between organisms in the way in which information flows from DNA to protein. Principal among these is that RNA transcripts in eukaryotic cells are subject to a series of processing steps in the nucleus, including RNA splicing, before they are permitted to exit from the nucleus and be translated into protein. As we discuss in this chapter, these processing steps can critically change the “meaning” of an RNA molecule and are therefore crucial for understanding how eukaryotic cells read their genome.


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Cell_Biology_Alberts. Although we focus on the production of the proteins encoded by the genome in this chapter, we see that for many genes, RNA is the final product. Like proteins, some of these RNAs fold into precise three-dimensional structures that have structural and catalytic roles in the cell. Other RNAs, as we discuss in the next chapter, act primarily as regulators of gene expression. But the roles of many non-coding RNAs are not yet known.


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Cell_Biology_Alberts. One might have predicted that the information present in genomes would be arranged in an orderly fashion, resembling a dictionary or a telephone directory. But it turns out that the genomes of most multicellular organisms are surprisingly disorderly, reflecting their chaotic evolutionary histories. The genes in these organisms largely consist of a long string of alternating short exons and long introns, as discussed in Chapter 4 (see Figure 4–15D). Moreover, small bits of DNA sequence that code for protein are interspersed with large blocks of seemingly meaningless DNA. Some sections of the genome contain many genes and others lack genes altogether. Proteins that work closely with one another in the cell often have their genes located on different chromosomes, and adjacent genes typically encode proteins that have little to do with each other in the cell. Decoding genomes is therefore no simple matter. Even with the aid of powerful computers, it is difficult for researchers to locate


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Cell_Biology_Alberts. encode proteins that have little to do with each other in the cell. Decoding genomes is therefore no simple matter. Even with the aid of powerful computers, it is difficult for researchers to locate definitively the beginning and end of genes, much less to decipher when and where each gene is expressed in the life of the