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Cell_Biology_Alberts. morphological stability of the lineage. However, comparisons between different present-day organisms show that the fraction of the genome subject to purifying selection is small; hence, it is fair to assume that the genome of the modern horseshoe crab, while preserving features critical for function, must differ greatly from that of its extinct ancestors, known to us only through the fossil record.


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Cell_Biology_Alberts. It is possible to get direct sequence information by examining DNA samples from ancient materials if these are not too old. In recent years, technical advances have allowed DNA sequencing from exceptionally well-preserved bone fragments that date from more than 100,000 years ago. Although any DNA this old will be imperfectly preserved, a sequence of the Neanderthal genome has been reconstructed from many millions of short DNA sequences, revealing—among other things—that our human ancestors interbred with Neanderthals in Europe and


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Cell_Biology_Alberts. Figure 4–69 A comparison of the β-globin gene cluster in the human and mouse genomes, showing the locations of transposable elements. This stretch of the human genome contains five functional β-globin-like genes (orange); the comparable region from the mouse genome has only four. The positions of the human Alu sequences are indicated by green circles, and the human L1 sequences by red circles. The mouse genome contains different but related transposable elements: the positions of b1 elements (which are related to the human Alu sequences) are indicated by blue triangles, and the positions of the mouse L1 elements (which are related to the human L1 sequences) are indicated by orange triangles. The absence of transposable elements from the globin structural genes can be attributed to purifying selection, which would have eliminated any insertion that compromised gene function. (Courtesy of Ross Hardison and webb miller.)


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Cell_Biology_Alberts. Figure 4–70 The puffer fish, Fugu rubripes. (Courtesy of byrappa venkatesh.) 0.0 100.0 180.0 thousands of nucleotide pairs that modern humans have inherited specific genes from them (Figure 4–72). The average difference in DNA sequence between humans and Neanderthals shows that our two lineages diverged somewhere between 270,000 and 440,000 years ago, well before the time that humans are believed to have migrated out of Africa.


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Cell_Biology_Alberts. But what about deciphering the genomes of much older ancestors, those for which no useful DNA samples can be isolated? For organisms that are as closely related as human and chimpanzee, we saw that this may not be difficult: reference to the gorilla sequence can be used to sort out which of the few sequence differences between human and chimpanzee are inherited from our common ancestor some 6 million years ago (see Figure 4–64). And for an ancestor that has produced a large number of different organisms alive today, the DNA sequences of many species can be compared simultaneously to unscramble much of the ancestral sequence, allowing scientists to derive DNA sequences much farther back in time. For example, from the genome sequences currently being obtained for dozens of modern placental mammals, it should be possible to infer much of the genome sequence of their 100 million-year-old common ancestor—the precursor of species as diverse as dog, mouse, rabbit, armadillo, and human (see


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Cell_Biology_Alberts. mammals, it should be possible to infer much of the genome sequence of their 100 million-year-old common ancestor—the precursor of species as diverse as dog, mouse, rabbit, armadillo, and human (see Figure 4–66).


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Cell_Biology_Alberts. multispecies Sequence Comparisons Identify Conserved DNA Sequences of Unknown Function The mass of DNA sequence now in databases (hundreds of billions of nucleotide pairs) provides a rich resource that scientists can mine for many purposes. This information can be used not only to unscramble the evolutionary pathways that have led to modern organisms, but also to provide insights into how cells and organisms function. Perhaps the most remarkable discovery in this realm comes from the observation that a striking amount of DNA sequence that does not code for protein has been conserved during mammalian evolution (see Table 4–1, p. 184). This is most clearly revealed when we align and compare DNA synteny


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Cell_Biology_Alberts. Figure 4–71 Comparison of the genomic sequences of the human and Fugu genes encoding the protein huntingtin. both genes (indicated in red) contain 67 short exons that align in 1:1 correspondence to one another; these exons are connected by curved lines. The human gene is 7.5 times larger than the Fugu gene (180,000 versus 24,000 nucleotide pairs). The size difference is entirely due to larger introns in the human gene. The larger size of the human introns is due in part to the presence of retrotransposons (discussed in Chapter 5), whose positions are represented by green vertical lines; the Fugu introns lack retrotransposons. In humans, mutation of the huntingtin gene causes Huntington’s disease, an inherited neurodegenerative disorder. (Adapted from S. baxendale et al., Nat. Genet. 10:67–76, 1995. with permission from macmillan Publishers ltd.)


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Cell_Biology_Alberts. Figure 4–72 The Neanderthals. (A) map of Europe showing the location of the cave in Croatia where most of the bones used to isolate the DNA used to derive the Neanderthal genome sequence were discovered. (b) Photograph of the vindija cave. (C) Photograph of the 38,000-yearold bones from vindija. more recent studies have succeeded in extracting DNA sequence information from hominid remains that are considerably older (see movie 8.3). (b, courtesy of Johannes krause; C, from R.E. green et al., Science 328: 710–722, 2010. Reprinted with permission from AAAS.) cave in Vindija, Croatia blocks from many different species, thereby identifying large numbers of so-called multispecies conserved sequences: some of these code for protein, but most of them do not (Figure 4–73).


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Cell_Biology_Alberts. Most of the noncoding conserved sequences discovered in this way turn out to be relatively short, containing between 50 and 200 nucleotide pairs. Among the most mysterious are the so-called “ultraconserved” noncoding sequences, exemplified by more than 5000 DNA segments over 100 nucleotides long that are exactly the same in human, mouse, and rat. Most have undergone little or no change since mammalian and bird ancestors diverged about 300 million years ago. The strict conservation implies that even though the sequences do not encode proteins, each nevertheless has an important function maintained by purifying selection. The puzzle is to unravel what those functions are.


Cell_Biology_Alberts

Cell_Biology_Alberts. Many of the conserved sequences that do not code for protein are now known to produce untranslated RNA molecules, such as the thousands of long noncoding RNAs (lncRNAs) that are thought to have important functions in regulating gene transcription. As we shall also see in Chapter 7, others are short regions of DNA scattered throughout the genome that directly bind proteins involved in gene regulation. But it is uncertain how much of the conserved noncoding DNA can be accounted for in these ways, and the function of most of it remains a mystery. This enigma highlights how much more we need to learn about the fundamental biological mechanisms that operate in animals and other complex organisms, and its solution is certain to have profound consequences for medicine.


Cell_Biology_Alberts

Cell_Biology_Alberts. How can cell biologists tackle the mystery of noncoding conserved DNA? Traditionally, attempts to determine the function of a puzzling DNA sequence begin by looking at the consequences of its experimental disruption. But many DNA sequences that are crucial for an organism in the wild can be expected to have no noticeable effect on its phenotype under laboratory conditions: what is required for a mouse to survive in a laboratory cage is very much less than what is required 190,000 nucleotide pairs 100 nucleotide pairs 10,000 nucleotide pairs Figure 4–73 The detection of multispecies conserved sequences.


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Cell_Biology_Alberts. In this example, genome sequences for each of the organisms shown have been compared with the indicated region of the human CFTR (cystic fibrosis transmembrane conductance regulator) gene; this region contains one exon plus a large amount of intronic DNA. For each organism, the percent identity with human for each 25-nucleotide block is plotted in green. In addition, a computational algorithm has been used to detect the sequences within this region that are most highly conserved when the sequences from all of the organisms are taken into account. besides the exon (dark blue on the line at the top of the figure), the positions of three other blocks of multispecies conserved sequences are indicated (pale blue). The function of most such sequences in the human genome is not known. (Courtesy of Eric D. green.) for it to succeed in nature. Moreover, calculations based on population genetics reveal that just a tiny selective advantage—less than a 0.1% difference in survival—can be enough to


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Cell_Biology_Alberts. Eric D. green.) for it to succeed in nature. Moreover, calculations based on population genetics reveal that just a tiny selective advantage—less than a 0.1% difference in survival—can be enough to strongly favor retaining a particular DNA sequence over evolutionary time spans. One should therefore not be surprised to find that many DNA sequences that are ultraconserved can be deleted from the mouse genome without any noticeable effect on that mouse in a laboratory.


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Cell_Biology_Alberts. A second important approach for discovering the function of a mysterious noncoding DNA sequence uses biochemical techniques to identify proteins or RNA molecules that bind to it—and/or to any RNA molecules that it produces. Most of this task still lies before us, but a start has been made (see p. 435).


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Cell_Biology_Alberts. Given genome sequence information, we can tackle another intriguing question: What alterations in our DNA have made humans so different from other animals—or for that matter, what makes any individual species so different from its relatives? For example, as soon as both the human and the chimpanzee genome sequences became available, scientists began searching for DNA sequence changes that might account for the striking differences between us and chimpanzees. With 3.2 billion nucleotide pairs to compare in the two species, this might seem an impossible task. But the job was made much easier by confining the search to 35,000 clearly defined multispecies conserved sequences (a total of about 5 million nucleotide pairs), representing parts of the genome that are most likely to be functionally important. Though these sequences are conserved strongly, they are not conserved perfectly, and when the version in one species is compared with that in another they are generally found to have


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Cell_Biology_Alberts. important. Though these sequences are conserved strongly, they are not conserved perfectly, and when the version in one species is compared with that in another they are generally found to have drifted apart by a small amount corresponding simply to the time elapsed since the last common ancestor. In a small proportion of cases, however, one sees signs of a sudden evolutionary spurt. For example, some DNA sequences that have been highly conserved in other mammalian species are found to have accumulated nucleotide changes exceptionally rapidly during the 6 million years of human evolution since we diverged from the chimpanzees. These human accelerated regions (HARs) are thought to reflect functions that have been especially important in making us different in some useful way.


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Cell_Biology_Alberts. About 50 such sites were identified in one study, one-fourth of which were located near genes associated with neural development. The sequence exhibiting the most rapid change (18 changes between human and chimpanzee, compared to only two changes between chimpanzee and chicken) was examined further and found to encode a 118-nucleotide noncoding RNA molecule, HAR1F (human accelerated region 1F), that is produced in the human cerebral cortex at a critical time during brain development. The function of this HAR1F RNA is not yet known, but findings of this type are stimulating research studies that may shed light on crucial features of the human brain.


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Cell_Biology_Alberts. A related approach in the search for the important mutations that contributed to human evolution likewise begins with DNA sequences that have been conserved during mammalian evolution, but rather than screening for accelerated changes in individual nucleotides, it focuses instead on chromosome sites that have experienced deletions in the 6 million years since our lineage diverged from that of chimpanzees. More than 500 such sequences—conserved among other species but deleted in humans—have been discovered. Each deletion removes an average of 95 nucleotides of DNA sequence. Only one of these deletions affects a protein-coding region: the rest are thought to alter regions that affect how nearby genes are expressed, an expectation that has been experimentally confirmed in a few cases. A large proportion of the presumed regulatory regions identified in this way lie near genes that affect neural function and/or near genes involved in steroid signaling, suggesting that changes in the


Cell_Biology_Alberts

Cell_Biology_Alberts. A large proportion of the presumed regulatory regions identified in this way lie near genes that affect neural function and/or near genes involved in steroid signaling, suggesting that changes in the nervous system and in immune or reproductive functions have played an especially important role in human evolution.


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Cell_Biology_Alberts. mutations in the DNA Sequences That Control gene Expression Have Driven many of the Evolutionary Changes in vertebrates


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Cell_Biology_Alberts. The vast hoard of genomic sequence data now being accumulated can be explored in many other ways to reveal events that happened even hundreds of millions of years ago. For example, one can attempt to trace the origins of the regulatory elements in DNA that have played critical parts in vertebrate evolution. One such study began with the identification of nearly 3 million noncoding sequences, averaging 28 base pairs in length, that have been conserved in recent vertebrate evolution while being absent in more ancient ancestors. Each of these special non-coding sequences is likely to represent a functional innovation peculiar to a particular branch of the vertebrate family tree, and most of them are thought to consist of regulatory DNA that governs the expression of a neighboring gene. Given full genome sequences, one can identify the genes that lie closest and thus appear most likely to have fallen under the sway of these novel regulatory elements. By comparing many different species,


Cell_Biology_Alberts

Cell_Biology_Alberts. full genome sequences, one can identify the genes that lie closest and thus appear most likely to have fallen under the sway of these novel regulatory elements. By comparing many different species, with known divergence times, one can also estimate when each such regulatory element came into existence as a conserved feature. The findings suggest remarkable evolutionary differences between the various functional classes of genes (Figure 4–74). Conserved regulatory elements that originated early in vertebrate evolution—that is, more than about 300 million years ago, which is when the mammalian lineage split from the lineage leading to birds and reptiles—seem to be mostly associated with genes that code for transcription regulator proteins and for proteins with roles in organizing embryonic development. Then came an era when the regulatory DNA innovations arose next to genes coding for receptors for extracellular signals. Finally, over the course of the past 100 million years, the


Cell_Biology_Alberts

Cell_Biology_Alberts. development. Then came an era when the regulatory DNA innovations arose next to genes coding for receptors for extracellular signals. Finally, over the course of the past 100 million years, the regulatory innovations seem to have been concentrated in the neighborhood of genes coding for proteins (such as protein kinases) that function to modify other proteins post-translationally.


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Cell_Biology_Alberts. Many questions remain to be answered about these phenomena and what they mean. One possible interpretation is that the logic—the circuit diagram—of the gene regulatory network in vertebrates was established early, and that more recent evolutionary change has mainly occurred through the tuning of quantitative parameters. This could help to explain why, among the mammals, for example, the basic body plan—the topology of the tissues and organs—has been largely conserved. gene Duplication Also Provides an Important Source of genetic Novelty During Evolution Evolution depends on the creation of new genes, as well as on the modification of those that already exist. How does this occur? When we compare organisms that seem very different—a primate with a rodent, for example, or a mouse with a fish—we rarely encounter genes in the one species that have no homolog in the reception of extracellular signals HUMAN 500 400 300 200 100 0 millions of years before present


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Cell_Biology_Alberts. Figure 4–74 The types of changes in gene regulation inferred to have predominated during the evolution of our vertebrate ancestors. To produce the information summarized in this plot, wherever possible the type of gene regulated by each conserved noncoding sequence was inferred from the identity of its closest protein-coding gene. The fixation time for each conserved sequence was then used to derive the conclusions shown. (based on C.b. lowe et al., Science 333:1019–1024, 2011. with permission from AAAS.) other. Genes without homologous counterparts are relatively scarce even when we compare such divergent organisms as a mammal and a worm. On the other hand, we frequently find gene families that have different numbers of members in different species. To create such families, genes have been repeatedly duplicated, and the copies have then diverged to take on new functions that often vary from one species to another.


Cell_Biology_Alberts

Cell_Biology_Alberts. Gene duplication occurs at high rates in all evolutionary lineages, contributing to the vigorous process of DNA addition discussed previously. In a detailed study of spontaneous duplications in yeast, duplications of 50,000 to 250,000 nucleotide pairs were commonly observed, most of which were tandemly repeated. These appeared to result from DNA replication errors that led to the inexact repair of double-strand chromosome breaks. A comparison of the human and chimpanzee genomes reveals that, since the time that these two organisms diverged, such segmental duplications have added about 5 million nucleotide pairs to each genome every million years, with an average duplication size being about 50,000 nucleotide pairs (although there are some duplications five times larger). In fact, if one counts nucleotides, duplication events have created more differences between our two species than have single-nucleotide substitutions.


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Cell_Biology_Alberts. What is the fate of newly duplicated genes? In most cases, there is presumed to be little or no selection—at least initially—to maintain the duplicated state since either copy can provide an equivalent function. Hence, many duplication events are likely to be followed by loss-of-function mutations in one or the other gene. This cycle would functionally restore the one-gene state that preceded the duplication. Indeed, there are many examples in contemporary genomes where one copy of a duplicated gene can be seen to have become irreversibly inactivated by multiple mutations. Over time, the sequence similarity between such a pseudogene and the functional gene whose duplication produced it would be expected to be eroded by the accumulation of many mutations in the pseudogene—the homologous relationship eventually becoming undetectable.


Cell_Biology_Alberts

Cell_Biology_Alberts. An alternative fate for gene duplications is for both copies to remain functional, while diverging in their sequence and pattern of expression, thus taking on different roles. This process of “duplication and divergence” almost certainly explains the presence of large families of genes with related functions in biologically complex organisms, and it is thought to play a critical role in the evolution of increased biological complexity. An examination of many different eukaryotic genomes suggests that the probability that any particular gene will undergo a duplication event that spreads to most or all individuals in a species is approximately 1 percent every million years.


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Cell_Biology_Alberts. Whole-genome duplications offer particularly dramatic examples of the duplication–divergence cycle. A whole-genome duplication can occur quite simply: all that is required is one round of genome replication in a germ-line cell lineage without a corresponding cell division. Initially, the chromosome number simply doubles. Such abrupt increases in the ploidy of an organism are common, particularly in fungi and plants. After a whole-genome duplication, all genes exist as duplicate copies. However, unless the duplication event occurred so recently that there has been little time for subsequent alterations in genome structure, the results of a series of segmental duplications—occurring at different times— are hard to distinguish from the end product of a whole-genome duplication. In mammals, for example, the role of whole-genome duplications versus a series of piecemeal duplications of DNA segments is quite uncertain. Nevertheless, it is clear that a great deal of gene duplication has


Cell_Biology_Alberts

Cell_Biology_Alberts. for example, the role of whole-genome duplications versus a series of piecemeal duplications of DNA segments is quite uncertain. Nevertheless, it is clear that a great deal of gene duplication has occurred in the distant past.


Cell_Biology_Alberts

Cell_Biology_Alberts. Analysis of the genome of the zebrafish, in which at least one whole-genome duplication is thought to have occurred hundreds of millions of years ago, has cast some light on the process of gene duplication and divergence. Although many duplicates of zebrafish genes appear to have been lost by mutation, a significant fraction—perhaps as many as 30–50%—have diverged functionally while both Figure 4–75 A comparison of the structure of one-chain and four-chain globins. The four-chain globin shown is hemoglobin, which is a complex of two α-globin and two β-globin chains. The one-chain globin present in some primitive vertebrates represents an intermediate in the evolution of the four-chain globin. with oxygen bound it exists as a monomer; without oxygen it dimerizes.


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Cell_Biology_Alberts. copies have remained active. In many cases, the most obvious functional difference between the duplicated genes is that they are expressed in different tissues or at different stages of development. One attractive theory to explain such an end result imagines that different, mildly deleterious mutations occur quickly in both copies of a duplicated gene set. For example, one copy might lose expression in a particular tissue as a result of a regulatory mutation, while the other copy loses expression in a second tissue. Following such an occurrence, both gene copies would be required to provide the full range of functions that were once supplied by a single gene; hence, both copies would now be protected from loss through inactivating mutations. Over a longer period, each copy could then undergo further changes through which it could acquire new, specialized features. The Evolution of the globin gene Family Shows How DNA Duplications Contribute to the Evolution of Organisms


Cell_Biology_Alberts

Cell_Biology_Alberts. The Evolution of the globin gene Family Shows How DNA Duplications Contribute to the Evolution of Organisms The globin gene family provides an especially good example of how DNA duplication generates new proteins, because its evolutionary history has been worked out particularly well. The unmistakable similarities in amino acid sequence and structure among the present-day globins indicate that they all must derive from a common ancestral gene, even though some are now encoded by widely separated genes in the mammalian genome.


Cell_Biology_Alberts

Cell_Biology_Alberts. We can reconstruct some of the past events that produced the various types of oxygen-carrying hemoglobin molecules by considering the different forms of the protein in organisms at different positions on the tree of life. A molecule like hemoglobin was necessary to allow multicellular animals to grow to a large size, since large animals cannot simply rely on the diffusion of oxygen through the body surface to oxygenate their tissues adequately. But oxygen plays a vital part in the life of nearly all living organisms, and oxygen-binding proteins homologous to hemoglobin can be recognized even in plants, fungi, and bacteria. In animals, the most primitive oxygen-carrying molecule is a globin polypeptide chain of about 150 amino acids that is found in many marine worms, insects, and primitive fish.


Cell_Biology_Alberts

Cell_Biology_Alberts. The hemoglobin molecule in more complex vertebrates, however, is composed of two kinds of globin chains. It appears that about 500 million years ago, during the continuing evolution of fish, a series of gene mutations and duplications occurred. These events established two slightly different globin genes in the genome of each individual, coding for αand β-globin chains that associate to form a hemoglobin molecule consisting of two α chains and two β chains (Figure 4–75). The four oxygen-binding sites in the α2β2 molecule interact, allowing a cooperative allosteric change in the molecule as it binds and releases oxygen, which enables hemoglobin to take up and release oxygen more efficiently than the single-chain version.


Cell_Biology_Alberts

Cell_Biology_Alberts. Still later, during the evolution of mammals, the β-chain gene apparently underwent duplication and mutation to give rise to a second β-like chain that millions of years ago is synthesized specifically in the fetus. The resulting hemoglobin molecule has a higher affinity for oxygen than adult hemoglobin and thus helps in the transfer of oxygen from the mother to the fetus. The gene for the new β-like chain subsequently duplicated and mutated again to produce two new genes, ε and γ, the ε chain being produced earlier in development (to form α2ε2) than the fetal γ chain, which forms α2γ2. A duplication of the adult β-chain gene occurred still later, during primate evolution, to give rise to a δ-globin gene and thus to a minor form of hemoglobin (α2δ2) that is found only in adult primates (Figure 4–76).


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Cell_Biology_Alberts. Each of these duplicated genes has been modified by point mutations that affect the properties of the final hemoglobin molecule, as well as by changes in regulatory regions that determine the timing and level of expression of the gene. Figure 4–76 An evolutionary scheme for the globin chains that carry oxygen in the blood of animals. The scheme emphasizes the β-like globin gene family. A relatively recent gene duplication of the γ-chain gene produced γg and γA, which are fetal β-like chains of identical function. The location of the globin genes in the human genome is shown at the top of the figure. As a result, each globin is made in different amounts at different times of human development.


Cell_Biology_Alberts

Cell_Biology_Alberts. As a result, each globin is made in different amounts at different times of human development. The history of these gene duplications is reflected in the arrangement of hemoglobin genes in the genome. In the human genome, the genes that arose from the original β gene are arranged as a series of homologous DNA sequences located within 50,000 nucleotide pairs of one another on a single chromosome. A similar cluster of human α-globin genes is located on a separate chromosome. Not only other mammals, but birds too have their αand β-globin gene clusters on separate chromosomes. In the frog Xenopus, however, they are together, suggesting that a chromosome translocation event in the lineage of birds and mammals separated the two gene clusters about 300 million years ago, soon after our ancestors diverged from amphibians (see Figure 4–76).


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Cell_Biology_Alberts. There are several duplicated globin DNA sequences in the αand β-globin gene clusters that are not functional genes but pseudogenes. These have a close sequence similarity to the functional genes but have been disabled by mutations that prevent their expression as functional proteins. The existence of such pseudogenes makes it clear that, as expected, not every DNA duplication leads to a new functional gene. genes Encoding New Proteins Can be Created by the Recombination of Exons


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Cell_Biology_Alberts. genes Encoding New Proteins Can be Created by the Recombination of Exons The role of DNA duplication in evolution is not confined to the expansion of gene families. It can also act on a smaller scale to create single genes by stringing together short duplicated segments of DNA. The proteins encoded by genes generated in this way can be recognized by the presence of repeating similar protein domains, which are covalently linked to one another in series. The immunoglobulins (Figure 4–77), for example, as well as most fibrous proteins (such as collagens) are encoded by genes that have evolved by repeated duplications of a primordial DNA sequence.


Cell_Biology_Alberts

Cell_Biology_Alberts. In genes that have evolved in this way, as well as in many other genes, each separate exon often encodes an individual protein folding unit, or domain. It is believed that the organization of DNA coding sequences as a series of such exons separated by long introns has greatly facilitated the evolution of new proteins. The duplications necessary to form a single gene coding for a protein with repeating domains, for example, can easily occur by breaking and rejoining the DNA anywhere in the long introns on either side of an exon; without introns there would be only a few sites in the original gene at which a recombinational exchange between DNA molecules could duplicate the domain and not disrupt it. By enabling the duplication to occur by recombination at many potential sites rather than just a few, introns increase the probability of a favorable duplication event.


Cell_Biology_Alberts

Cell_Biology_Alberts. More generally, we know from genome sequences that the various parts of genes—both their individual exons and their regulatory elements—have served as modular elements that have been duplicated and moved about the genome to create the great diversity of living things. Thus, for example, many present-day proteins are formed as a patchwork of domains from different origins, reflecting their complex evolutionary history (see Figure 3–17). Neutral mutations Often Spread to become Fixed in a Population, with a Probability That Depends on Population Size In comparisons between two species that have diverged from one another by millions of years, it makes little difference which individuals from each species are Figure 4–77 Schematic view of an antibody (immunoglobulin) molecule.


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Cell_Biology_Alberts. Figure 4–77 Schematic view of an antibody (immunoglobulin) molecule. This molecule is a complex of two identical heavy chains and two identical light chains. Each heavy chain contains four similar, covalently linked domains, while each light chain contains two such domains. Each domain is encoded by a separate exon, and all of the exons are thought to have evolved by the serial duplication of a single ancestral exon.


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Cell_Biology_Alberts. compared. For example, typical human and chimpanzee DNA sequences differ from one another by about 1%. In contrast, when the same region of the genome is sampled from two randomly chosen humans, the differences are typically about 0.1%. For more distantly related organisms, the interspecies differences outshine intraspecies variation even more dramatically. However, each “fixed difference” between the human and the chimpanzee (in other words, each difference that is now characteristic of all or nearly all individuals of each species) started out as a new mutation in a single individual. If the size of the interbreeding population in which the mutation occurred is N, the initial allele frequency for a new mutation would be 1/(2N) for a diploid organism. How does such a rare mutation become fixed in the population, and hence become a characteristic of the species rather than of a few scattered individuals?


Cell_Biology_Alberts

Cell_Biology_Alberts. The answer to this question depends on the functional consequences of the mutation. If the mutation has a significantly deleterious effect, it will simply be eliminated by purifying selection and will not become fixed. (In the most extreme case, the individual carrying the mutation will die without producing progeny.) Conversely, the rare mutations that confer a major reproductive advantage on individuals who inherit them can spread rapidly in the population. Because humans reproduce sexually and genetic recombination occurs each time a gamete is formed (discussed in Chapter 5), the genome of each individual who has inherited the mutation will be a unique recombinational mosaic of segments inherited from a large number of ancestors. The selected mutation along with a modest amount of neighboring sequence—ultimately inherited from the individual in which the mutation occurred—will simply be one piece of this huge mosaic.


Cell_Biology_Alberts

Cell_Biology_Alberts. The great majority of mutations that are not harmful are not beneficial either. These selectively neutral mutations can also spread and become fixed in a population, and they make a large contribution to evolutionary change in genomes. For example, as we saw earlier, they account for most of the DNA sequence differences between apes and humans. The spread of neutral mutations is not as rapid as the spread of the rare strongly advantageous mutations. It depends on a random variation in the number of mutation-bearing progeny produced by each mutation-bearing individual, causing changes in the relative frequency of the mutant allele in the population. Through a sort of “random walk” process, the mutant allele may eventually become extinct, or it may become commonplace. This can be modeled mathematically for an idealized interbreeding population, on the assumption of constant population size and random mating, as well as selective neutrality for the mutations. While neither of the first


Cell_Biology_Alberts

Cell_Biology_Alberts. for an idealized interbreeding population, on the assumption of constant population size and random mating, as well as selective neutrality for the mutations. While neither of the first two assumptions is a good description of human population history, study of this idealized case reveals the general principles in a clear and simple way.


Cell_Biology_Alberts

Cell_Biology_Alberts. When a new neutral mutation occurs in a population of constant size N that is undergoing random mating, the probability that it will ultimately become fixed is approximately 1/(2N). This is because there are 2N copies of the gene in the diploid population, and each of them has an equal chance of becoming the predominant version in the long run. For those mutations that do become fixed, the mathematics shows that the average time to fixation is approximately 4N generations. Detailed analyses of data on human genetic variation have suggested an ancestral population size of approximately 10,000 at the time when the current pattern of genetic variation was largely established. With a population that has reached this size, the probability that a new, selectively neutral mutation would become fixed is small (1/20,000), while the average time to fixation would be on the order of 800,000 years (assuming a 20-year generation time). Thus, while we know that the human population has grown


Cell_Biology_Alberts

Cell_Biology_Alberts. fixed is small (1/20,000), while the average time to fixation would be on the order of 800,000 years (assuming a 20-year generation time). Thus, while we know that the human population has grown enormously since the development of agriculture approximately 15,000 years ago, most of the present-day set of common human genetic variants reflects the mixture of variants that was already present long before this time, when the human population was still small.


Cell_Biology_Alberts

Cell_Biology_Alberts. Similar arguments explain another phenomenon with important practical implications for genetic counseling. In an isolated community descended from a small group of founders, such as the people of Iceland or the Jews of Eastern individual with rare allele Europe, genetic variants that are rare in the human population as a whole can often be present at a high frequency, even if those variants are mildly deleterious (Figure 4–78). A great Deal Can be learned from Analyses of the variation Among Humans Even though the common variant gene alleles among modern humans originate from variants present in a comparatively tiny group of ancestors, the total number of variants now encountered, including those that are individually rare, is very large. New neutral mutations are constantly occurring and accumulating, even though no single one of them has had enough time to become fixed in the vast modern human population.


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Cell_Biology_Alberts. From detailed comparisons of the DNA sequences of a large number of modern humans located around the globe, scientists can estimate how many generations have elapsed since the origin of a particular neutral mutation. From such data, it has been possible to map the routes of ancient human migrations. For example, by combining this type of genetic analysis with archaeological findings, scientists have been able to deduce the most probable routes that our ancestors took when they left Africa 60,000 to 80,000 years ago (Figure 4–79).


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Cell_Biology_Alberts. We have been focusing on mutations that affect a single gene, but these are not the only source of variation. Another source, perhaps even more important but missed for many years, lies in the many duplications and deletions of large blocks of human DNA. When one compares any individual human with the standard reference genome in the database, one will generally find roughly 100 differences involving gain or loss of long sequence blocks, totaling perhaps 3 million nucleotide pairs. Some of these copy number variations (CNVs) will be very common, presumably reflecting relatively ancient origins, while others will be present in only a small minority of people (Figure 4–80). On average, nearly half of the CNVs contain known genes. CNVs have been implicated in many human traits, including color blindness, infertility, hypertension, and a wide variety of disease susceptibilities. In retrospect, this type of variation is not surprising, given the prominent role of DNA addition and DNA loss


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Cell_Biology_Alberts. blindness, infertility, hypertension, and a wide variety of disease susceptibilities. In retrospect, this type of variation is not surprising, given the prominent role of DNA addition and DNA loss in vertebrate evolution.


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Cell_Biology_Alberts. The intraspecies variations that have been most extensively characterized, however, are single-nucleotide polymorphisms (SNPs). These are simply points in the genome sequence where one large fraction of the human population has one nucleotide, while another substantial fraction has another. To qualify as Figure 4–78 How founder effects determine the set of genetic variants in a population of individuals belonging to the same species. This example illustrates how a rare allele (red) can become established in an isolated population, even though the mutation that produced it has no selective advantage—or is mildly deleterious.


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Cell_Biology_Alberts. Figure 4–79 Tracing the course of human history by analyses of genome sequences. The map shows the routes of the earliest successful human migrations. Dotted lines indicate two alternative routes that our ancestors are thought to have taken out of Africa. DNA sequence comparisons suggest that modern Europeans descended from a small ancestral population that existed about 30,000 to 50,000 years ago. In agreement, archaeological findings suggest that the ancestors of modern native Australians (solid red arrows)—and of modern European and middle Eastern populations—reached their destinations about 45,000 years ago. Even more recent studies, comparing the genome sequences of living humans with those of Neanderthals and another extinct population from southern Siberia (the Denisovans), suggest that our exit from Africa was a bit more convoluted, while also revealing that a number of our ancestors interbred with these hominid neighbors as they made their way across the globe. (modified from


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Cell_Biology_Alberts. P. Forster and S. matsumura, Science 308:965–966, 2005.) density of known genes a polymorphism, the variants must be common enough to give a reasonably high probability that the genomes of two randomly chosen individuals will differ at the given site; a probability of 1% is commonly chosen as the cutoff. Two human genomes sampled from the modern world population at random will differ at approximately 2.5 × 106 such sites (1 per 1300 nucleotide pairs). As will be described in the overview of genetics in Chapter 8, SNPs in the human genome can be extremely useful for genetic mapping analyses, in which one attempts to associate specific traits (phenotypes) with specific DNA sequences for medical or scientific purposes (see p. 493). But while useful as genetic markers, there is good evidence that most of these SNPs have little or no effect on human fitness. This is as expected, since deleterious variants will have been selected against during human evolution and, unlike SNPs, should


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Cell_Biology_Alberts. that most of these SNPs have little or no effect on human fitness. This is as expected, since deleterious variants will have been selected against during human evolution and, unlike SNPs, should therefore be rare.


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Cell_Biology_Alberts. Against the background of ordinary SNPs inherited from our prehistoric ancestors, certain sequences with exceptionally high mutation rates stand out. A dramatic example is provided by CA repeats, which are ubiquitous in the human genome and in the genomes of other eukaryotes. Sequences with the motif (CA)n are replicated with relatively low fidelity because of a slippage that occurs between the template and the newly synthesized strands during DNA replication; hence, the precise value of n can vary over a considerable range from one genome to the next. These repeats make ideal DNA-based genetic markers, since most humans are heterozygous, having inherited one repeat length (n) from their mother and a different repeat length from their father. While the value of n changes sufficiently rarely that most parent–child transmissions propagate CA repeats faithfully, the changes are sufficiently frequent to maintain high levels of heterozygosity in the human population. These and some other


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Cell_Biology_Alberts. that most parent–child transmissions propagate CA repeats faithfully, the changes are sufficiently frequent to maintain high levels of heterozygosity in the human population. These and some other simple repeats that display exceptionally high variability therefore provide the basis for identifying individuals by DNA analysis in crime investigations, paternity suits, and other forensic applications (see Figure 8–39).


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Cell_Biology_Alberts. While most of the SNPs and CNVs in the human genome sequence are thought to have little or no effect on phenotype, a subset of the genome sequence variations must be responsible for the heritable aspects of human individuality. We know that even a single nucleotide change that alters one amino acid in a protein can cause a serious disease, as for example in sickle-cell anemia, which is caused by such a mutation in hemoglobin (Movie 4.3). We also know that gene dosage—a doubling or halving of the copy number of some genes—can have a profound effect on development by altering the level of gene product, as can changes in regulatory DNA sequences. There is therefore every reason to suppose that some of the many differences between any two human beings will have substantial Figure 4–80 Detection of copy number variations on human chromosome 17.


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Cell_Biology_Alberts. when 100 individuals were tested by a DNA microarray analysis that detects the copy number of DNA sequences throughout the entire length of this chromosome, the indicated distributions of DNA additions (green bars) and DNA losses (red bars) were observed compared with an arbitrary human reference sequence. The shortest red and green bars represent a single occurrence among the 200 chromosomes examined, whereas the longer bars indicate that the addition or loss was correspondingly more frequent. The results show preferred regions where the variations occur, and these tend to be in or near regions that already contain blocks of segmental duplications. many of the changes include known genes. (Adapted from J.l. Freeman et al., Genome Res. 16:949–961, 2006. with permission from Cold Spring Harbor laboratory Press.) effects on human health, physiology, behavior, and physique. A major challenge in human genetics is to recognize those relatively few variations that are functionally important


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Cell_Biology_Alberts. laboratory Press.) effects on human health, physiology, behavior, and physique. A major challenge in human genetics is to recognize those relatively few variations that are functionally important against a large background of variation that is neutral and of no consequence.


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Cell_Biology_Alberts. Comparisons of the nucleotide sequences of present-day genomes have revolutionized our understanding of gene and genome evolution. Because of the extremely high fidelity of DNA replication and DNA repair processes, random errors in maintaining the nucleotide sequences in genomes occur so rarely that only about one nucleotide in a thousand is altered in every million years in any particular eukaryotic line of descent. Not surprisingly, therefore, a comparison of human and chimpanzee chromosomes—which are separated by about 6 million years of evolution— reveals very few changes. Not only are our genes essentially the same, but their order on each chromosome is almost identical. Although a substantial number of segmental duplications and segmental deletions have occurred in the past 6 million years, even the positions of the transposable elements that make up a major portion of our noncoding DNA are mostly unchanged.


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Cell_Biology_Alberts. When one compares the genomes of two more distantly related organisms—such as a human and a mouse, separated by about 80 million years—one finds many more changes. Now the effects of natural selection can be clearly seen: through purifying selection, essential nucleotide sequences—both in regulatory regions and in coding sequences (exons)—have been highly conserved. In contrast, nonessential sequences (for example, much of the DNA in introns) have been altered to such an extent that one can no longer see any family resemblance.


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Cell_Biology_Alberts. Because of purifying selection, the comparison of the genome sequences of multiple related species is an especially powerful way to find DNA sequences with important functions. Although about 5% of the human genome has been conserved as a result of purifying selection, the function of the majority of this DNA (tens of thousands of multispecies conserved sequences) remains mysterious. Future experiments characterizing its functions should teach us many new lessons about vertebrate biology.


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Cell_Biology_Alberts. Other sequence comparisons show that a great deal of the genetic complexity of present-day organisms is due to the expansion of ancient gene families. DNA duplication followed by sequence divergence has clearly been a major source of genetic novelty during evolution. On a more recent time scale, the genomes of any two humans will differ from each other both because of nucleotide substitutions (single-nucleotide polymorphisms, or SNPs) and because of inherited DNA gains and DNA losses that cause copy number variations (CNVs). Understanding the effects of these differences will improve both medicine and our understanding of human biology. Which statements are true? explain why or why not. How many different types of chromatin structure are important for cells? How is each of these structures established and maintained, and which ones tend to be inherited following DNA replication?


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Cell_Biology_Alberts. How many different types of chromatin structure are important for cells? How is each of these structures established and maintained, and which ones tend to be inherited following DNA replication? why are there so many different chromatin remodeling complexes in cells? what are their essential roles, and how do they get loaded onto chromatin at specific places and at specific times? How do chromosomal loops form during interphase, and what happens to these loops in condensed mitotic chromosomes? what genetic changes made us uniquely human? what further aspects of our recent evolutionary development can be reconstructed by sequencing DNA from remains of ancient hominids? How much of the enormous complexity that we find in cell biology is unnecessary, having evolved by random drift? 4–1 Human females have 23 different chromosomes, served DNA sequences facilitates the search for function-whereas human males have 24. ally important regions.


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Cell_Biology_Alberts. 4–1 Human females have 23 different chromosomes, served DNA sequences facilitates the search for function-whereas human males have 24. ally important regions. 4–2 The four core histones are relatively small proteins 4–5 Gene duplication and divergence is thought to with a very high proportion of positively charged amino have played a critical role in the evolution of increased bioacids; the positive charge helps the histones bind tightly to logical complexity. DNA, regardless of its nucleotide sequence. Discuss the following problems. 4–3 Nucleosomes bind DNA so tightly that they cannot 4–6 DNA isolated from the bacterial virus M13 con-move from the positions where they are first assembled. tains 25% A, 33% T, 22% C, and 20% G. Do these results 4–4 In a comparison between the DNAs of related strike you as peculiar? Why or why not? How might you organisms such as humans and mice, identifying the con-explain these values?


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Cell_Biology_Alberts. Figure Q4–1 Three nucleotides from the interior of a single strand of DNA (Problem 4–7). Arrows O at the ends of the DNA strand indicate that the structure continues in both directions. 4–7 A segment of DNA from the interior of a single strand is shown in O Figure Q4–1. What is the polarity of this –O P O DNA from top to bottom? O on a molar basis. What are the mole percents of A, G, and T? in the human lineage (Figure Q4–2). Draw the intermediate chromosome that resulted from the first inversion and explicitly indicate the segments O included in each inversion. (Problem 4–9). Differently colored blocks indicate segments of the chromosomes that are homologous in DNA sequence.


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Cell_Biology_Alberts. (Problem 4–9). Differently colored blocks indicate segments of the chromosomes that are homologous in DNA sequence. 4–10 Assuming that the 30-nm chromatin fiber contains about 20 nucleosomes (200 bp/nucleosome) per 50 nm of length, calculate the degree of compaction of DNA associated with this level of chromatin structure. What fraction of the 10,000-fold condensation that occurs at mitosis does this level of DNA packing represent? 4–11 In contrast to histone acetylation, which always correlates with gene activation, histone methylation can lead to either transcriptional activation or repression. How do you suppose that the same modification—methylation—can mediate different biological outcomes?


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Cell_Biology_Alberts. 4–12 Why is a chromosome with two centromeres (a dicentric chromosome) unstable? Would a backup centromere not be a good thing for a chromosome, giving it two chances to form a kinetochore and attach to microtubules during mitosis? Would that not help to ensure that the chromosome did not get left behind at mitosis? 4–13 Look at the two yeast colonies in Figure Q4–3. Each of these colonies contains about 100,000 cells descended from a single yeast cell, originally somewhere in the middle of the clump. A white colony arises when the Ade2 gene is expressed from its normal chromosomal location. When the Ade2 gene is moved to a location near a telomere, it is packed into heterochromatin and inactivated in most cells, giving rise to colonies that are mostly red. In these largely red colonies, white sectors fan out from the middle of the colony. In both the red and white sectors, the Ade2 white colony of yeast cells red colony of yeast cells with white sectors


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Cell_Biology_Alberts. Figure Q4–3 Position effect on expression of the yeast Ade2 gene (Problem 4–13). The Ade2 gene codes for one of the enzymes of adenosine biosynthesis, and the absence of the Ade2 gene product leads to the accumulation of a red pigment. Therefore a colony of cells that express Ade2 is white, and one composed of cells in which the Ade2 gene is not expressed is red. gene is still located near telomeres. Explain why white sectors have formed near the rim of the red colony. Based on the patterns observed, what can you conclude about the propagation of the transcriptional state of the Ade2 gene from mother to daughter cells in this experiment?


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Cell_Biology_Alberts. 4–14 Mobile pieces of DNA—transposable elements— that insert themselves into chromosomes and accumulate during evolution make up more than 40% of the human genome. Transposable elements of four types—long interspersed nuclear elements (LINEs), short interspersed nuclear elements (SINEs), long terminal repeat (LTR) retrotransposons, and DNA transposons—are inserted more-or-less randomly throughout the human genome. These elements are conspicuously rare at the four homeobox gene clusters, HoxA, HoxB, HoxC, and HoxD, as illustrated for HoxD in Figure Q4–4, along with an equivalent region of chromosome 22, which lacks a Hox cluster. Each Hox cluster is about 100 kb in length and contains 9 to 11 genes, whose differential expression along the anteroposterior axis of the developing embryo establishes the basic body plan for humans (and for other animals). Why do you suppose that transposable elements are so rare in the Hox clusters?


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Cell_Biology_Alberts. Figure Q4–4 Transposable elements and genes in 1-mb regions of chromosomes 2 and 22 (Problem 4–14). Blue lines that project upward indicate exons of known genes. Red lines that project downward indicate transposable elements; they are so numerous (constituting more than 40% of the human genome) that they merge into nearly a solid block outside the Hox clusters. (Adapted from E. lander et al., Nature 409:860–921, 2001. with permission from macmillan Publishers ltd.) Armstrong l (2014) Epigenetics. New York: garland Science. Hartwell l, Hood l, goldberg ml et al. (2010) genetics: From genes to genomes, 4th ed. boston, mA: mcgraw Hill. Jobling m, Hollox E, Hurles m et al. (2014) Human Evolutionary genetics, 2nd ed. New York: garland Science. Strachan T & Read AP (2010) Human molecular genetics, 4th ed. New York: garland Science. The Structure and Function of DNA


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Cell_Biology_Alberts. Strachan T & Read AP (2010) Human molecular genetics, 4th ed. New York: garland Science. The Structure and Function of DNA Avery OT, macleod Cm & mcCarty m (1944) Studies on the chemical nature of the substance inducing transformation of pneumococcal types. J. Exp. Med. 79, 137–158. meselson m & Stahl Fw (1958) The replication of DNA in Escherichia coli. Proc. Natl Acad. Sci. USA 44, 671–682. watson JD & Crick FHC (1953) molecular structure of nucleic acids. A structure for deoxyribose nucleic acid. Nature 171, 737–738. Chromosomal DNA and Its Packaging in the Chromatin Fiber Andrews AJ & luger k (2011) Nucleosome structure(s) and stability: variations on a theme. Annu. Rev. Biophys. 40, 99–117. Avvakumov N, Nourani A & Cõté J (2011) Histone chaperones: modulators of chromatin marks. Mol. Cell 41, 502–514. Deal Rb, Henikoff Jg & Henikoff S (2010) genome-wide kinetics of nucleosome turnover determined by metabolic labeling of histones. Science 328, 1161–1164.


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Cell_Biology_Alberts. Deal Rb, Henikoff Jg & Henikoff S (2010) genome-wide kinetics of nucleosome turnover determined by metabolic labeling of histones. Science 328, 1161–1164. grigoryev SA & woodcock Cl (2012) Chromatin organization—the 30 nm fiber. Exp. Cell Res. 318, 1448–1455. li g, levitus m, bustamante C & widom J (2005) Rapid spontaneous accessibility of nucleosomal DNA. Nat. Struct. Mol. Biol. 12, 46–53. luger k, mäder Aw, Richmond Rk et al. (1997) Crystal structure of the nucleosome core particle at 2.8 Å resolution. Nature 389, 251–260. Narlikar gJ, Sundaramoorthy R & Owen-Hughes T (2013) mechanisms and functions of ATP-dependent chromatin-remodeling enzymes. Cell 154, 490–503. Song F, Chen P, Sun D et al. (2014) Cryo-Em study of the chromatin fiber reveals a double helix twisted by tetranucleosomal units. Science 344, 376–380.


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Cell_Biology_Alberts. Song F, Chen P, Sun D et al. (2014) Cryo-Em study of the chromatin fiber reveals a double helix twisted by tetranucleosomal units. Science 344, 376–380. Al-Sady b, madhani HD & Narlikar gJ (2013) Division of labor between the chromodomains of HP1 and Suv39 methylase enables coordination of heterochromatin spread. Mol. Cell 51, 80–91. beisel C & Paro R (2011) Silencing chromatin: comparing modes and mechanisms. Nat. Rev. Genet. 12, 123–135. black bE, Jansen lET, Foltz DR & Cleveland Dw (2011) Centromere identity, function, and epigenetic propagation across cell divisions. Cold Spring Harb. Symp. Quant. Biol. 75, 403–418. Elgin SCR & Reuter g (2013) Position-effect variegation, heterochromatin formation, and gene silencing in Drosophila. Cold Spring Harb. Perspect. Biol. 5, a017780. Felsenfeld g (2014) A brief history of epigenetics. Cold Spring Harb. Perspect. Biol. 6, a018200.


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Cell_Biology_Alberts. Felsenfeld g (2014) A brief history of epigenetics. Cold Spring Harb. Perspect. Biol. 6, a018200. Feng S, Jacobsen SE & Reik w (2010) Epigenetic reprogramming in plant and animal development. Science 330, 622–627. Filion gJ, van bemmel Jg, braunschweig U et al. (2010) Systematic protein location mapping reveals five principal chromatin types in Drosophila cells. Cell 143, 212–224. Fodor bD, Shukeir N, Reuter g & Jenuwein T (2010) mammalian Su(var) genes in chromatin control. Annu. Rev. Cell Dev. Biol. 26, 471–501. giles kE, gowher H, ghirlando R et al. (2010) Chromatin boundaries, insulators, and long-range interactions in the nucleus. Cold Spring Harb. Symp. Quant. Biol. 75, 79–85. gohl D, Aoki T, blanton J et al. (2011) mechanism of chromosomal boundary action: roadblock, sink, or loop? Genetics 187, 731–748. mellone b, Erhardt S & karpen gH (2006) The AbCs of centromeres. Nat. Cell Biol. 8, 427–429.


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Cell_Biology_Alberts. mellone b, Erhardt S & karpen gH (2006) The AbCs of centromeres. Nat. Cell Biol. 8, 427–429. morris SA, baek S, Sung m-H et al. (2014) Overlapping chromatin-remodeling systems collaborate genome wide at dynamic chromatin transitions. Nat. Struct. Mol. Biol. 21, 73–81. Politz JCR, Scalzo D & groudine m (2013) Something silent this way forms: the functional organization of the repressive nuclear compartment. Annu. Rev. Cell Dev. Biol. 29, 241–270. Rothbart Sb & Strahl bD (2014) Interpreting the language of histone and DNA modifications. Biochim. Biophys. Acta 1839, 627–643. weber Cm & Henikoff S (2014) Histone variants: dynamic punctuation in transcription. Genes Dev. 28, 672–682. Xu m, long C, Chen X et al. (2010) Partitioning of histone H3-H4 tetramers during DNA replication-dependent chromatin assembly. Science 328, 94–98.


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Cell_Biology_Alberts. Xu m, long C, Chen X et al. (2010) Partitioning of histone H3-H4 tetramers during DNA replication-dependent chromatin assembly. Science 328, 94–98. The Global Structure of Chromosomes belmont AS (2014) large-scale chromatin organization: the good, the surprising, and the still perplexing. Curr. Opin. Cell Biol. 26, 69–78. bickmore w (2013) The spatial organization of the human genome. Annu. Rev. Genomics Hum. Genet. 14, 67–84. Callan Hg (1982) lampbrush chromosomes. Proc. R. Soc. Lond. B Biol. Sci. 214, 417–448. Cheutin T, bantignies F, leblanc b & Cavalli g (2010) Chromatin folding: from linear chromosomes to the 4D nucleus. Cold Spring Harb. Symp. Quant. Biol. 75, 461–473. Cremer T & Cremer m (2010) Chromosome territories. Cold Spring Harb. Perspect. Biol. 2, a003889. lieberman-Aiden E, van berkum Nl, williams l et al. (2009) Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science 326, 289–293.


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Cell_Biology_Alberts. lieberman-Aiden E, van berkum Nl, williams l et al. (2009) Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science 326, 289–293. maeshima k & laemmli Uk (2003) A two-step scaffolding model for mitotic chromosome assembly. Dev. Cell 4, 467–480. moser SC & Swedlow JR (2011) How to be a mitotic chromosome. Chromosome Res. 19, 307–319. Nizami ZF, Deryusheva S & gall Jg (2010) Cajal bodies and histone locus bodies in Drosophila and Xenopus. Cold Spring Harb. Symp. Quant. Biol. 75, 313–320. Zhimulev IF (1997) Polytene chromosomes, heterochromatin, and position effect variegation. Adv. Genet. 37, 1–566. batzer mA & Deininger Pl (2002) Alu repeats and human genomic diversity. Nat. Rev. Genet. 3, 370–379. Feuk l, Carson AR & Scherer S (2006) Structural variation in the human genome. Nat. Rev. Genet. 7, 85–97. green RE, krause J, briggs Aw et al. (2010) A draft sequence of the Neandertal genome. Science 328, 710–722.


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Cell_Biology_Alberts. green RE, krause J, briggs Aw et al. (2010) A draft sequence of the Neandertal genome. Science 328, 710–722. International Human genome Sequencing Consortium (2001) Initial sequencing and analysis of the human genome. Nature 409, 860–921. International Human genome Sequencing Consortium (2004) Finishing the euchromatic sequence of the human genome. Nature 431, 931–945. kellis m, wold b, Snyder mP et al. (2014) Defining functional DNA elements in the human genome. Proc. Natl Acad. Sci. USA 111, 6131–6138. lander ES (2011) Initial impact of the sequencing of the human genome. Nature 470, 187–197. lee C & Scherer Sw (2010) The clinical context of copy number variation in the human genome. Expert Rev. Mol. Med. 12, e8. mouse genome Sequencing Consortium (2002) Initial sequencing and comparative analysis of the mouse genome. Nature 420, 520–562.


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Cell_Biology_Alberts. mouse genome Sequencing Consortium (2002) Initial sequencing and comparative analysis of the mouse genome. Nature 420, 520–562. Pollard kS, Salama SR, lambert N et al. (2006) An RNA gene expressed during cortical development evolved rapidly in humans. Nature 443, 167–172. DNA Replication, Repair, and Recombination


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Cell_Biology_Alberts. DNA Replication, Repair, and Recombination The ability of cells to maintain a high degree of order in a chaotic universe depends upon the accurate duplication of vast quantities of genetic information carried in chemical form as DNA. This process, called DNA replication, must occur before a cell can produce two genetically identical daughter cells. Maintaining order also requires the continued surveillance and repair of this genetic information, because DNA inside cells is repeatedly damaged by chemicals and radiation from the environment, as well as by thermal accidents and reactive molecules generated inside the cell. In this chapter, we describe the protein machines that replicate and repair the cell’s DNA. These machines catalyze some of the most rapid and accurate processes that take place within cells, and their mechanisms illustrate the elegance and efficiency of cell chemistry.


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Cell_Biology_Alberts. While the short-term survival of a cell can depend on preventing changes in its DNA, the long-term survival of a species requires that DNA sequences be changeable over many generations to permit evolutionary adaptation to changing circumstances. We shall see that despite the great efforts that cells make to protect their DNA, occasional changes in DNA sequences do occur. Over time, these changes provide the genetic variation upon which selection pressures act during the evolution of organisms. We begin this chapter with a brief discussion of the changes that occur in DNA as it is passed down from generation to generation. Next, we discuss the cell mechanisms—DNA replication and DNA repair—that are responsible for minimizing these changes. Finally, we consider some of the most intriguing pathways that alter DNA sequences—in particular, those of DNA recombination including the movement within chromosomes of special DNA sequences called transposable elements.


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Cell_Biology_Alberts. Although, as just pointed out, occasional genetic changes enhance the long-term survival of a species through evolution, the survival of the individual demands a high degree of genetic stability. Only rarely do the cell’s DNA-maintenance processes fail, resulting in permanent change in the DNA. Such a change is called a mutation, and it can destroy an organism if it occurs in a vital position in the DNA sequence.


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Cell_Biology_Alberts. The mutation rate, the rate at which changes occur in DNA sequences, can be determined directly from experiments carried out with a bacterium such as Escherichia coli—a resident of our intestinal tract and a commonly used laboratory organism (see Figure 1–24). Under laboratory conditions, E. coli divides about once every 30 minutes, and a single cell can generate a very large population— several billion—in less than a day. In such a population, it is possible to detect the small fraction of bacteria that have suffered a damaging mutation in a particular gene, if that gene is not required for the bacterium’s survival. For example, the mutation rate of a gene specifically required for cells to use the sugar lactose as an energy source can be determined by growing the cells in the presence of a different sugar, such as glucose, and testing them subsequently to see how many have lost the ability to survive on a lactose diet. The fraction of damaged genes underestimates the actual mutation


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Cell_Biology_Alberts. a different sugar, such as glucose, and testing them subsequently to see how many have lost the ability to survive on a lactose diet. The fraction of damaged genes underestimates the actual mutation rate because many mutations are silent (for example, those that change a codon but not the amino acid it specifies, or those that change an amino acid without affecting the activity of the protein coded for by the gene). After correcting for these silent mutations, one finds that a single gene that encodes an average-sized protein (~103 coding nucleotide pairs) accumulates a mutation (not necessarily one that would inactivate the protein) approximately once in about 106 bacterial cell generations. Stated differently, bacteria display a mutation rate of about three nucleotide changes per 1010 nucleotides per cell generation.


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Cell_Biology_Alberts. Recently, it has become possible to measure the germ-line mutation rate directly in more complex, sexually reproducing organisms such as humans. In this case, the complete genomes from a family—parents and offspring—were directly sequenced, and a careful comparison revealed that approximately 70 new single-nucleotide mutations arose in the germ lines of each offspring. Normalized to the size of the human genome, the mutation rate is one nucleotide change per 108 nucleotides per human generation. This is a slight underestimate because some mutations will be lethal and will therefore be absent from progeny; however, because relatively little of the human genome carries critical information, this consideration has only a small effect on the true mutation rate. It is estimated that approximately 100 cell divisions occur in the germ line from the time of conception to the time of production of the eggs and sperm that go on to make the next generation. Thus, the human mutation rate,


Cell_Biology_Alberts

Cell_Biology_Alberts. 100 cell divisions occur in the germ line from the time of conception to the time of production of the eggs and sperm that go on to make the next generation. Thus, the human mutation rate, expressed in terms of cell divisions (instead of human generations), is approximately 1 mutation/1010 nucleotides/ cell division.


Cell_Biology_Alberts

Cell_Biology_Alberts. Although E. coli and humans differ greatly in their modes of reproduction and in their generation times, when the mutation rates of each are normalized to a single round of DNA replication, they are both extremely low and within a factor of three of each other. We shall see later in the chapter that the basic mechanisms that ensure these low rates of mutation have been conserved since the very early history of cells on Earth. Low Mutation Rates Are Necessary for Life as We Know It


Cell_Biology_Alberts

Cell_Biology_Alberts. Low Mutation Rates Are Necessary for Life as We Know It Since many mutations are deleterious, no species can afford to allow them to accumulate at a high rate in its germ cells. Although the observed mutation frequency is low, it is nevertheless thought to limit the number of essential proteins that any organism can depend upon to perhaps 30,000. More than this, and the probability that at least one critical component will suffer a damaging mutation becomes catastrophically high. By an extension of the same argument, a mutation frequency tenfold higher would limit an organism to about 3000 essential genes. In this case, evolution would have been limited to organisms considerably less complex than a fruit fly.


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Cell_Biology_Alberts. The cells of a sexually reproducing animal or plant are of two types: germ cells and somatic cells. The germ cells transmit genetic information from parent to offspring; the somatic cells form the body of the organism (Figure 5–1). We have seen that germ cells must be protected against high rates of mutation to maintain the species. However, the somatic cells of multicellular organisms must also be protected from genetic change to properly maintain the organized structure of the body. Nucleotide changes in somatic cells can give rise to variant cells, some of which, through “local” natural selection, proliferate rapidly at the expense of the rest of the organism. In an extreme case, the result is the uncontrolled cell proliferation that we know as cancer, a disease that causes (in Europe and North America) more than 20% of human deaths each year. These deaths are due largely to an accumulation of changes in the DNA sequences of somatic cells, as discussed in Chapter 20. A significant


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Cell_Biology_Alberts. and North America) more than 20% of human deaths each year. These deaths are due largely to an accumulation of changes in the DNA sequences of somatic cells, as discussed in Chapter 20. A significant increase in the mutation frequency would presumably cause a disastrous increase in the incidence of cancer by accelerating the rate at which somatic-cell variants arise. Thus, both for the perpetuation of a species with a large number of genes (germ-cell stability) and for the prevention of cancer resulting from mutations in somatic cells (somatic-cell stability), multicellular organisms like ourselves depend on the remarkably high fidelity with which their DNA sequences are replicated and maintained.


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Cell_Biology_Alberts. In all cells, DNA sequences are maintained and replicated with high fidelity. The mutation rate, approximately one nucleotide change per 1010 nucleotides each time the DNA is replicated, is roughly the same for organisms as different as bacteria and humans. Because of this remarkable accuracy, the sequence of the human genome (approximately 3.2 × 109 nucleotide pairs) is unchanged or changed by only a few nucleotides each time a typical human cell divides. This allows most humans to pass accurate genetic instructions from one generation to the next, and also to avoid the changes in somatic cells that lead to cancer. All organisms duplicate their DNA with extraordinary accuracy before each cell division. In this section, we explore how an elaborate “replication machine” achieves this accuracy, while duplicating DNA at rates as high as 1000 nucleotides per second.


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Cell_Biology_Alberts. As introduced in Chapter 1, DNA templating is the mechanism the cell uses to copy the nucleotide sequence of one DNA strand into a complementary DNA sequence (Figure 5–2). This process requires the separation of the DNA helix into two template strands, and entails the recognition of each nucleotide in the DNA template strands by a free (unpolymerized) complementary nucleotide. The separation of Figure 5–1 Germ-line cells and somatic cells carry out fundamentally different functions. In sexually reproducing organisms, the germ-line cells (red) propagate genetic information into the next generation. Somatic cells (blue), which form the body of the organism, are necessary for the survival of germ-line cells but do not themselves leave any progeny. Figure 5–2 The DNA double helix acts as a template for its own duplication.


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Cell_Biology_Alberts. Figure 5–2 The DNA double helix acts as a template for its own duplication. Because the nucleotide A will pair successfully only with T, and G only with C, each strand of DNA can serve as a template to specify the sequence of nucleotides in its complementary strand by DNA base-pairing. In this way, a double-helical DNA molecule can be copied precisely. the DNA helix exposes the hydrogen-bond donor and acceptor groups on each DNA base for base-pairing with the appropriate incoming free nucleotide, aligning it for its enzyme-catalyzed polymerization into a new DNA chain. The first nucleotide-polymerizing enzyme, DNA polymerase, was discovered in 1957. The free nucleotides that serve as substrates for this enzyme were found to be deoxyribonucleoside triphosphates, and their polymerization into DNA required a single-strand DNA template. Figure 5–3 and Figure 5–4 illustrate the stepwise mechanism of this reaction. The DNA Replication Fork Is Asymmetrical


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Cell_Biology_Alberts. The DNA Replication Fork Is Asymmetrical During DNA replication inside a cell, each of the two original DNA strands serves as a template for the formation of an entire new strand. Because each of the two daughters of a dividing cell inherits a new DNA double helix containing one original and one new strand (Figure 5–5), the DNA double helix is said to be replicated “semiconservatively.” How is this feat accomplished? 3˜ end of strand 5˜ end of strand Figure 5–3 The chemistry of DNA synthesis. The addition of a deoxyribonucleotide to the 3ʹ end of a polynucleotide chain (the primer strand) is the fundamental reaction by which DNA is synthesized. As shown, base-pairing between an incoming deoxyribonucleoside triphosphate and an existing strand of DNA (the template strand) guides the formation of the new strand of DNA and causes it to have a complementary nucleotide sequence. The way in which complementary nucleotides base-pair is shown in Figure 4–4. direction of chain growth


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Cell_Biology_Alberts. Figure 5–4 DNA synthesis catalyzed by DNA polymerase. (A) DNA polymerase catalyzes the stepwise addition of a deoxyribonucleotide to the 3ʹ-OH end of a polynucleotide chain, the growing primer strand that is paired to an existing template strand. The newly synthesized DNA strand therefore polymerizes in the 5ʹ-to-3ʹ direction as shown also in the previous figure. Because each incoming deoxyribonucleoside triphosphate must pair with the template strand to be recognized by the DNA polymerase, this strand determines which of the four possible deoxyribonucleotides (A, C, G, or T) will be added. The reaction is driven by a large, favorable free-energy change, caused by the release of pyrophosphate and its subsequent hydrolysis to two molecules of inorganic phosphate. (B) Structure of DNA polymerase complexed wth DNA (orange), as determined by x-ray crystallography (Movie 5.1). The template DNA strand is the longer strand and the newly synthesized DNA is the shorter. (C) Schematic diagram


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Cell_Biology_Alberts. complexed wth DNA (orange), as determined by x-ray crystallography (Movie 5.1). The template DNA strand is the longer strand and the newly synthesized DNA is the shorter. (C) Schematic diagram of DNA polymerase, based on the structure in (B). The proper base-pair geometry of a correct incoming deoxyribonucleoside triphosphate causes the polymerase to tighten around the base pair, thereby initiating the nucleotide addition reaction (middle diagram (C)). Dissociation of pyrophosphate relaxes the polymerase, allowing translocation of the DNA by one nucleotide so the active site of the polymerase is ready to receive the next deoxyribonucleoside triphosphate.


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Cell_Biology_Alberts. Analyses carried out in the early 1960s on whole replicating chromosomes revealed a localized region of replication that moves progressively along the parental DNA double helix. Because of its Y-shaped structure, this active region is called a replication fork (Figure 5–6). At the replication fork, a multienzyme complex that contains the DNA polymerase synthesizes the DNA of both new daughter strands.


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Cell_Biology_Alberts. Initially, the simplest mechanism of DNA replication seemed to be the continuous growth of both new strands, nucleotide by nucleotide, at the replication fork as it moves from one end of a DNA molecule to the other. But because of the antiparallel orientation of the two DNA strands in the DNA double helix (see Figure 5–2), this mechanism would require one daughter strand to polymerize in the 5ʹ-to-3ʹ direction and the other in the 3ʹ-to-5ʹ direction. Such a replication fork would require two distinct types of DNA polymerase enzymes. However, as attractive as this model might be, the DNA polymerases at replication forks can synthesize only in the 5ʹ-to-3ʹ direction.


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Cell_Biology_Alberts. How, then, can a DNA strand grow in the 3ʹ-to-5ʹ direction? The answer was first suggested by the results of an experiment performed in the late 1960s. Researchers added highly radioactive 3H-thymidine to dividing bacteria for a few seconds, so that only the most recently replicated DNA—that just behind the replication fork—became radiolabeled. This experiment revealed the transient existence of pieces of DNA that were 1000–2000 nucleotides long, now commonly known as Okazaki fragments, at the growing replication fork. (Similar replication Figure 5–5 The semiconservative nature of DNA replication. In a round of replication, each of the two strands of DNA is used as a template for the formation of a complementary DNA strand. The original strands therefore remain intact through many cell generations.


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Cell_Biology_Alberts. intermediates were later found in eukaryotes, where they are only 100–200 nucleotides long.) The Okazaki fragments were shown to be polymerized only in the 5ʹ-to-3ʹ chain direction and to be joined together after their synthesis to create long DNA chains. A replication fork therefore has an asymmetric structure (Figure 5–7). The DNA daughter strand that is synthesized continuously is known as the leading strand. Its synthesis slightly precedes the synthesis of the daughter strand that is synthesized discontinuously, known as the lagging strand. For the lagging strand, the direction of nucleotide polymerization is opposite to the overall direction of DNA chain growth. The synthesis of this strand by a discontinuous “backstitching” mechanism means that DNA replication requires only the 5ʹ-to-3ʹ type of DNA polymerase. The High Fidelity of DNA Replication Requires Several Proofreading Mechanisms


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Cell_Biology_Alberts. The High Fidelity of DNA Replication Requires Several Proofreading Mechanisms As discussed above, the fidelity of copying DNA during replication is such that only about one mistake occurs for every 1010 nucleotides copied. This fidelity is much higher than one would expect from the accuracy of complementary base-pairing. The standard complementary base pairs (see Figure 4–4) are not the only ones possible. For example, with small changes in helix geometry, two hydrogen bonds can form between G and T in DNA. In addition, rare tautomeric forms of the four DNA bases occur transiently in ratios of 1 part to 104 or 105. These forms mispair without a change in helix geometry: the rare tautomeric form of C pairs with A instead of G, for example.


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Cell_Biology_Alberts. If the DNA polymerase did nothing special when a mispairing occurred between an incoming deoxyribonucleoside triphosphate and the DNA template, the wrong nucleotide would often be incorporated into the new DNA chain, producing frequent mutations. The high fidelity of DNA replication, however, depends not only on the initial base-pairing but also on several “proofreading” mechanisms that act sequentially to correct any initial mispairings that might have occurred. Figure 5–6 Two replication forks moving in opposite directions on a circular chromosome. An active zone of DNA replication moves progressively along a replicating DNA molecule, creating a Y-shaped DNA structure known as a replication fork: the two arms of each Y are the two daughter DNA molecules, and the stem of the Y is the parental DNA helix. In this diagram, parental strands are orange; newly synthesized strands are red. 1 µm (Micrograph courtesy of Jerome Vinograd.)


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Cell_Biology_Alberts. Figure 5–7 The structure of a DNA replication fork. Left, replication fork with newly synthesized DNA in red and arrows indicating the 5ʹ-to-3ʹ direction of DNA synthesis. Because both daughter DNA strands are polymerized in the 5ʹ-to-3ʹ direction, the DNA synthesized on the lagging strand must be made initially as a series of short DNA molecules, called Okazaki fragments, named after the scientist who discovered them. Right, the same fork a short time later. On the lagging strand, the Okazaki fragments are synthesized sequentially, with those nearest the fork being the most recently made.


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Cell_Biology_Alberts. DNA polymerase performs the first proofreading step just before a new nucleotide is covalently added to the growing chain. Our knowledge of this mechanism comes from studies of several different DNA polymerases, including one produced by a bacterial virus, T7, that replicates inside E. coli. The correct nucleotide has a higher affinity for the moving polymerase than does the incorrect nucleotide, because the correct pairing is more energetically favorable. Moreover, after nucleotide binding, but before the nucleotide is covalently added to the grow-AAAAAAAAA ing chain, the enzyme must undergo a conformational change in which its “grip” tightens around the active site (see Figure 5–4). Because this change occurs more readily with correct than incorrect base-pairing, it allows the polymerase to “double-check” the exact base-pair geometry before it catalyzes the addition of the nucleotide. Incorrectly paired nucleotides are harder to add and therefore more likely to diffuse away before


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Cell_Biology_Alberts. to “double-check” the exact base-pair geometry before it catalyzes the addition of the nucleotide. Incorrectly paired nucleotides are harder to add and therefore more likely to diffuse away before the polymerase can mistakenly add them.


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Cell_Biology_Alberts. The next error-correcting reaction, known as exonucleolytic proofreading, takes place immediately after those rare instances in which an incorrect nucle otide is covalently added to the growing chain. DNA polymerase enzymes are unpaired or mispaired residues at the primer terminus, continuing until enough nucleotides have been removed to regenerate a correctly base-paired 3ʹ-OH ter minus that can prime DNA synthesis. In this way, DNA polymerase functions as a attached to DNA polymerase “self-correcting” enzyme that removes its own polymerization errors as it moves chews back to create a base- along the DNA (Figure 5–8 and Figure 5–9). paired 3˜-OH end on the primer


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Cell_Biology_Alberts. paired 3˜-OH end on the primer The self-correcting properties of the DNA polymerase depend on its require ment for a perfectly base-paired primer terminus, and it is apparently not possible for such an enzyme to start synthesis de novo, without an existing primer. By contrast, the RNA polymerase enzymes involved in gene transcription do not need such an efficient exonucleolytic proofreading mechanism: errors in making RNA are not passed on to the next generation, and the occasional defective RNA molecule that is produced has no long-term significance. RNA polymerases are thus able to start new polynucleotide chains without a primer. DNA polymerase resumes the process of adding nucleotides to the base-paired 3˜-OH end of the primer strand


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Cell_Biology_Alberts. DNA polymerase resumes the process of adding nucleotides to the base-paired 3˜-OH end of the primer strand Figure 5–8 Exonucleolytic proofreading by DNA polymerase during DNA replication. In this example, a C is accidentally incorporated at the growing 3ʹ-OH end of a DNA chain. The part of DNA polymerase that removes the misincorporated nucleotide is a specialized member of a large class of enzymes, known as exonucleases, that cleave nucleotides one at a time from the ends of polynucleotides. Figure 5–9 Editing by DNA polymerase. DNA polymerase complexed with the DNA template in the polymerizing mode (left) and the editing mode (right). The catalytic sites for the exonucleolytic (E) and the polymerization (P) reactions are indicated. In the editing mode, the newly synthesized DNA transiently unpairs from the template and enters the editing site where the most recently added nucleotide is catalytically removed.


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Cell_Biology_Alberts. There is an error frequency of about one mistake for every 104 polymerization events both in RNA synthesis and in the separate process of translating mRNA sequences into protein sequences. This error rate is over 100,000 times greater than that in DNA replication, where, as we have seen, a series of proofreading processes makes the process unusually accurate (Table 5–1). Only DNA Replication in the 5ʹ-to-3ʹ Direction Allows Efficient Error Correction


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Cell_Biology_Alberts. Only DNA Replication in the 5ʹ-to-3ʹ Direction Allows Efficient Error Correction The need for accuracy probably explains why DNA replication occurs only in the 5ʹ-to-3ʹ direction. If there were a DNA polymerase that added deoxyribonucleoside triphosphates in the 3ʹ-to-5ʹ direction, the growing 5ʹ end of the chain, rather than the incoming mononucleotide, would have to provide the activating triphosphate needed for the covalent linkage. In this case, the mistakes in polymerization could not be simply hydrolyzed away, because the bare 5ʹ end of the chain thus created would immediately terminate DNA synthesis (see Figure 5–3). It is therefore possible to correct a mismatched base only if it has been added to the 3ʹ end of a DNA chain. Although the backstitching mechanism for DNA replication seems complex, it preserves the 5ʹ-to-3ʹ direction of polymerization that is required for exonucleolytic proofreading.


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Cell_Biology_Alberts. Despite these safeguards against DNA replication errors, DNA polymerases occasionally make mistakes. However, as we shall see later, cells have yet another Figure 5–10 RNA primer synthesis. A schematic view of the reaction catalyzed by DNA primase, the enzyme that synthesizes the short RNA primers made on the lagging strand using DNA as a template. Unlike DNA polymerase, this enzyme can start a new polynucleotide chain by joining two nucleoside triphosphates together. The primase synthesizes a short polynucleotide in the 5ʹ-to-3ʹ direction and then stops, making the 3ʹ end of this primer available for the DNA polymerase. chance to correct these errors by a process called strand-directed mismatch repair. Before discussing this mechanism, however, we describe the other types of proteins that function at the replication fork. A Special Nucleotide-Polymerizing Enzyme Synthesizes Short RNA Primer Molecules on the Lagging Strand


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Cell_Biology_Alberts. A Special Nucleotide-Polymerizing Enzyme Synthesizes Short RNA Primer Molecules on the Lagging Strand For the leading strand, a primer is needed only at the start of replication: once a replication fork is established, the DNA polymerase is continuously presented with a base-paired chain end on which to add new nucleotides. On the lagging side of the fork, however, each time the DNA polymerase completes a short DNA Okazaki fragment (which takes a few seconds), it must start synthesizing a completely new fragment at a site further along the template strand (see Figure 5–7). A special mechanism produces the base-paired primer strand required by the DNA polymerase molecules. The mechanism depends on an enzyme called DNA primase, which uses ribonucleoside triphosphates to synthesize short RNA primers on the lagging strand (Figure 5–10). In eukaryotes, these primers are about 10 nucleotides long and are made at intervals of 100–200 nucleotides on the lagging strand.


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Cell_Biology_Alberts. The chemical structure of RNA was introduced in Chapter 1 and is described in detail in Chapter 6. Here, we note only that RNA is very similar in structure to DNA. A strand of RNA can form base pairs with a strand of DNA, generating a DNA–RNA hybrid double helix if the two nucleotide sequences are complementary. Thus, the same templating principle used for DNA synthesis guides the synthesis of RNA primers. Because an RNA primer contains a properly base-paired nucleotide with a 3ʹ-OH group at one end, it can be elongated by the DNA polymerase at this end to begin an Okazaki fragment. The synthesis of each Okazaki fragment ends when this DNA polymerase runs into the RNA primer attached to the 5ʹ end of the previous fragment. To produce a continuous DNA chain from the many DNA fragments made on the lagging strand, a special DNA repair system acts quickly to erase the old RNA primer and replace it with DNA. An enzyme called DNA ligase then joins the 3ʹend of the new DNA fragment to the


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Cell_Biology_Alberts. on the lagging strand, a special DNA repair system acts quickly to erase the old RNA primer and replace it with DNA. An enzyme called DNA ligase then joins the 3ʹend of the new DNA fragment to the 5ʹend of the previous one to complete the process (Figure 5–11 and Figure 5–12).


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Cell_Biology_Alberts. Why might an erasable RNA primer be preferred to a DNA primer that would not need to be erased? The argument that a self-correcting polymerase cannot start chains de novo also implies the converse: an enzyme that starts chains anew cannot be efficient at self-correction. Thus, any enzyme that primes the synthesis of Okazaki fragments will of necessity make a relatively inaccurate copy (at least one error in 105). Even if the copies retained in the final product constituted as little as 5% of the total genome (for example, 10 nucleotides per 200-nucleotide DNA fragment), the resulting increase in the overall mutation rate would be enormous. It therefore seems likely that the use of RNA rather than DNA for priming brings a powerful advantage to the cell: the ribonucleotides in the primer automatically mark these sequences as “suspect copy” to be efficiently removed and replaced.


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Cell_Biology_Alberts. Figure 5–11 The synthesis of one of many DNA fragments on the lagging strand. In eukaryotes, RNA primers are made at intervals spaced by about 200 nucleotides on the lagging strand, and each RNA primer is approximately 10 nucleotides long. This primer is erased by a special DNA repair enzyme (an RNAse H) that recognizes an RNA strand in an RNA/DNA helix and fragments it; this leaves gaps that are filled in by DNA polymerase and DNA ligase. DNA polymerase adds to new RNA primer to start new Okazaki fragment sealing by DNA ligase joins new Okazaki fragment to the growing chain Special Proteins Help to Open Up the DNA Double Helix in Front of the Replication Fork


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Cell_Biology_Alberts. Special Proteins Help to Open Up the DNA Double Helix in Front of the Replication Fork For DNA synthesis to proceed, the DNA double helix must be opened up (“melted”) ahead of the replication fork so that the incoming deoxyribonucleoside triphosphates can form base pairs with the template strands. However, the DNA double helix is very stable under physiological conditions; the base pairs are locked in place so strongly that it requires temperatures approaching that of boiling water to separate the two strands in a test tube. For this reason, two additional types of replication proteins—DNA helicases and single-strand DNA-binding proteins—are needed to open the double helix and provide the appropriate single-strand DNA templates for the DNA polymerase to copy.


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Cell_Biology_Alberts. DNA helicases were first isolated as proteins that hydrolyze ATP when they are bound to single strands of DNA. As described in Chapter 3, the hydrolysis of ATP can change the shape of a protein molecule in a cyclical manner that allows the protein to perform mechanical work. DNA helicases use this principle to propel themselves rapidly along a DNA single strand. When they encounter a region of double helix, they continue to move along their strand, thereby prying apart the helix at rates of up to 1000 nucleotide pairs per second (Figure 5–13 and Figure 5–14). The two strands of DNA have opposite polarities, and, in principle, a helicase


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Cell_Biology_Alberts. The two strands of DNA have opposite polarities, and, in principle, a helicase Figure 5–12 The reaction catalyzed by DNA ligase. This enzyme seals a broken phosphodiester bond. As shown, DNA ligase uses a molecule of ATP to activate the 5ʹ end at the nick (step 1) before forming the new bond (step 2). In this way, the energetically unfavorable nick-sealing reaction is driven by being coupled to the energetically favorable process of ATP hydrolysis. could unwind the DNA double helix by moving in the 5ʹ-to-3ʹ direction along one 5˜ 3˜ strand or in the 3ʹ-to-5ʹ direction along the other. In fact, both types of DNA helicase exist. In the best-understood replication systems in bacteria, a helicase moving 5ʹ to 3ʹ along the lagging-strand template appears to have the predominant role, for reasons that will become clear shortly.


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Cell_Biology_Alberts. Single-strand DNA-binding (SSB) proteins, also called helix-destabilizing proteins, bind tightly and cooperatively to exposed single-strand DNA without covering the bases, which therefore remain available as templates. These proteins are unable to open a long DNA helix directly, but they aid helicases by stabilizing the unwound, single-strand conformation. In addition, through cooperative binding, they coat and straighten out the regions of single-strand DNA, which occur routinely in the lagging-strand template, thereby preventing the formation of the short hairpin helices that readily form in single-strand DNA (Figure 5–15 and Figure 5–16). If not removed, these hairpin helices can impede the DNA synthesis catalyzed by DNA polymerase. A Sliding Ring Holds a Moving DNA Polymerase Onto the DNA


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Cell_Biology_Alberts. A Sliding Ring Holds a Moving DNA Polymerase Onto the DNA On their own, most DNA polymerase molecules will synthesize only a short string of nucleotides before falling off the DNA template. The tendency to dissociate quickly from a DNA molecule allows a DNA polymerase molecule that has just Figure 5–13 An assay for DNA helicase enzymes. A short DNA fragment is annealed to a long DNA single strand to form a region of DNA double helix. The double helix is melted as the helicase runs along the DNA single strand, releasing the short DNA fragment in a reaction that requires the presence of both the helicase protein and ATP. The rapid stepwise movement of the helicase is powered by its ATP hydrolysis (shown schematically in Figure 3–75A). As indicated, many DNA helicases are composed of six subunits.