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AP1-mediated Multidrug Resistance in Saccharomyces cerevisiae Requires FLR1 Encoding a Transporter of the Major Facilitator Superfamily*

We have isolated a Candida albicansgene that confers resistance to the azole derivative fluconazole (FCZ) when overexpressed in Saccharomyces cerevisiae. This gene encodes a protein highly homologous to S. cerevisiae yAP-1, a bZip transcription factor known to mediate cellular resistance to toxicants such as cycloheximide (CYH), 4-nitroquinolineN-oxide (4-NQO), cadmium, and hydrogen peroxide. The gene was named CAP1, for C. albicansAP-1 . Cap1 and yAP-1 are functional homologues, since CAP1 expression in a yap1mutant strain partially restores the ability of the cells to grow on toxic concentrations of cadmium or hydrogen peroxide. We have found that the expression of YBR008c, an open reading frame identified in the yeast genome sequencing project and predicted to code for a multidrug transporter of the major facilitator superfamily, is dramatically induced in S. cerevisiae cells overexpressingCAP1. Overexpression of either CAP1 orYAP1 in a wild-type strain results in resistance to FCZ, CYH, and 4-NQO, whereas such resistance is completely abrogated (FCZ and CYH) or strongly reduced (4-NQO) in a ybr008c deletion mutant, demonstrating that YBR008c is involved inYAP1- and CAP1-mediated multidrug resistance.YBR008c has been renamed FLR1, forfluconazole resistance 1. The expression of an FLR1-lacZ reporter construct is strongly induced by the overexpression of either CAP1 orYAP1, indicating that the FLR1 gene is transcriptionally regulated by the Cap1 and yAP-1 proteins. Taken collectively, our results demonstrate that FLR1 represents a new YAP1-controlled multidrug resistance molecular determinant in S. cerevisiae. A similar detoxification pathway is also likely to operate in C. albicans.

We have isolated a Candida albicans gene that confers resistance to the azole derivative fluconazole (FCZ) when overexpressed in Saccharomyces cerevisiae. This gene encodes a protein highly homologous to S. cerevisiae yAP-1, a bZip transcription factor known to mediate cellular resistance to toxicants such as cycloheximide (CYH), 4-nitroquinoline N-oxide (4-NQO), cadmium, and hydrogen peroxide. The gene was named CAP1, for C. albicans AP-1. Cap1 and yAP-1 are functional homologues, since CAP1 expression in a yap1 mutant strain partially restores the ability of the cells to grow on toxic concentrations of cadmium or hydrogen peroxide. We have found that the expression of YBR008c, an open reading frame identified in the yeast genome sequencing project and predicted to code for a multidrug transporter of the major facilitator superfamily, is dramatically induced in S. cerevisiae cells overexpressing CAP1. Overexpression of either CAP1 or YAP1 in a wild-type strain results in resistance to FCZ, CYH, and 4-NQO, whereas such resistance is completely abrogated (FCZ and CYH) or strongly reduced (4-NQO) in a ybr008c deletion mutant, demonstrating that YBR008c is involved in YAP1-and CAP1-mediated multidrug resistance. YBR008c has been renamed FLR1, for fluconazole resistance 1. The expression of an FLR1-lacZ reporter construct is strongly induced by the overexpression of either CAP1 or YAP1, indicating that the FLR1 gene is transcriptionally regulated by the Cap1 and yAP-1 proteins. Taken collectively, our results demonstrate that FLR1 represents a new YAP1-controlled multidrug resistance molecular determinant in S. cerevisiae. A similar detoxification pathway is also likely to operate in C. albicans.
Cells have evolved elaborate molecular mechanisms to protect themselves from injuries caused by environmental exposure to toxic compounds of different structures and functions.
One of these mechanisms, termed multidrug resistance (MDR) 1 or pleiotropic drug resistance (PDR), operates in a wide variety of cell types, including bacteria, protozoans, fungi, and mammalian cells (1)(2)(3)(4). It involves a network of membrane-associated transporters acting as multidrug efflux pumps and transcription factors regulating the expression of these pumps.
Transcription factors such as Pdr1, Pdr3, and yAP-1 are also implicated in MDR (23)(24)(25). Pdr1 and Pdr3, two homologous zinc finger proteins belonging to the Zn 2 C 6 binuclear zinc cluster family, have been shown to control the expression of the ABC transporters PDR5, SNQ2, and YOR1 (23, 24, 26 -29). yAP-1 (also known as Pdr4/Snq3/Par1) is also associated with resistance to a variety of toxicants, including CYH, 4-NQO, sulfometuron methyl, and cadmium, and with tolerance to oxidative stress induced by compounds such as hydrogen peroxide and diamide (30 -37). yAP-1 belongs to a family of basic domain-leucine zipper (bZip) transcription factors, which includes S. cerevisiae Gcn4 and mammalian Fos and Jun (25). It binds to a conserved DNA sequence termed the yAP-1 response element (YRE) within the promoter of its targets to activate their transcription (34). yAP-1 targets include the cadmium resistance ABC transporter gene YCF1 (15,16), as well as a number of genes involved in response to oxidative stress such as GSH1, encoding ␥-glutamylcysteine synthase (38); TRX2, one of the two genes coding for thioredoxin (36); and GLR1 encoding glutathione reductase (39). A yAP-1 homologue, Cad1/yAP-2, has been isolated based on its ability to confer resistance to cadmium and to 1,10-phenanthroline, but the target genes mediating this resistance are still unknown (33,40).
Candida albicans is an opportunistic yeast that causes severe infections in immunocompromised individuals (41). Among the different agents employed in antifungal therapy, the azole derivative FCZ is the most widely used because of its low toxicity and its high efficacy (42). However, the successful treatment of candidosis by FCZ has been impaired by the emergence of drug resistant strains in patients undergoing long term or prophylactic treatment, mostly AIDS patients (42)(43)(44). A number of studies investigating the mechanisms of FCZ resistance in C. albicans and other Candida species have shown that resistant strains fail to accumulate FCZ due to an increased drug efflux, suggesting the participation of transporter-mediated drug resistance mechanisms in these strains (45)(46)(47)(48)(49). Two C. albicans genes coding for transporters of the ABC superfamily, CDR1 and CDR2, have been recently isolated by complementation of an S. cerevisiae pdr5 null mutant (50,51). In addition, the C. albicans BEN r gene, coding for a multidrug transporter of the MF superfamily, has been cloned through its ability to confer benomyl resistance when overexpressed in S. cerevisiae (52,53). CDR1, CDR2, and BEN r have been shown to be overexpressed in C. albicans clinical and experimental FCZ-resistant isolates (47,48,51). However, the molecular mechanisms controlling the transcriptional induction of these transporters in response to drug exposure have not yet been elucidated.
In this report, we describe the identification of a new molecular determinant of C. albicans FCZ resistance by functional overexpression in S. cerevisiae. This gene, named CAP1, codes for a bZip transcription factor structurally and functionally related to S. cerevisiae yAP-1 (25). A search for the transcriptional target(s) of Cap1 mediating FCZ resistance in S. cerevisiae has identified FLR1, predicted to code for a transporter of the MF superfamily highly homologous to C. albicans BEN r , as being an MDR effector of both Cap1 and yAP-1 (5,7). This is the first demonstration that yeast AP-1 proteins can confer MDR by transcriptionally regulating the expression of a transporter of the MF superfamily.
Stock Solutions of Antifungal Compounds-Stock solutions of FCZ (provided by Pfizer Canada Inc., Arnprior, Ontario) and CYH (Sigma, Mississauga, Ontario) were prepared in water at 10 mg/ml. Stock solutions of 4-NQO (Sigma) were prepared in dimethyl sulfoxide at 5 mM. Cadmium sulfate (MAT Laboratory, Beauport, Québec) was dissolved in water at 50 mM. Stocks were aliquoted and kept at Ϫ80°C.
Isolation of the CAP1 Gene-A YEp352-based C. albicans genomic DNA library (a gift from C. Boone, Simon Fraser University, Burnaby, British Columbia) was introduced in W303 cells (56). YEp352 is a URA3-based, multicopy vector (57). Ura ϩ transformants were selected on SD Ϫura plates, harvested, and plated on YPD plates containing FCZ at 500 g/ml. Plasmids from resistant colonies were recovered, analyzed by restriction mapping, and found to carry identical genomic DNA inserts. Plasmid pF1 was chosen for further characterization. Secondary pF1 transformants were found to be resistant to FCZ, thus confirming the plasmid dependence of the resistance phenotype. A 3-kb SmaI-PstI fragment from pF1 was subcloned in plasmid YEp352 cleaved with SmaI and PstI, to give plasmid pF1a. DNA sequence analysis of pF1a revealed that it contained a truncated ORF. To obtain a full-length clone, a C. albicans fosmid library was screened with a pF1a subfragment as probe, yielding five positive fosmids (B. Magee, University of Minnesota, St. Paul, MN). A 3.2-kb BglII fosmid fragment hybridizing with the probe was further subcloned into plasmid YEp352 at the BamHI site, yielding plasmid FosBgl.
DNA Sequencing and Analysis-Sequencing of the CAP1 gene was performed on both DNA strands of the entire 3.2-kb BglII genomic DNA fragment with custom synthesized oligonucleotides, using the automated sequencing facilities of the Sheldon Biotechnology Center (McGill University, Montreal, Canada). Sequence analyses were performed using the University of Wisconsin Genetics Computer Group programs (58).
CAP1 and YAP1 Expression Plasmids-The FosBgl and pF1a plasmids were used throughout this study for expression of the full-length and truncated versions of the CAP1 gene and were renamed YEp352/ CAP1 and YEp352/CAP1-TR, respectively. A 2.5-kb DNA fragment overlapping the entire YAP1 gene (positions Ϫ349 to ϩ2129 with respect to the initiation codon) (25) was amplified by polymerase chain reaction (PCR) using S. cerevisiae genomic DNA as template, the highfidelity Pfu DNA polymerase (Stratagene), and oligonucleotides 5Ј-CGGATTCTAGCTGTTTGGTTGAT and 5Ј-CGGAATTCATACATAG-TCTA. The resulting PCR fragment was gel-purified and cloned blunt into YEp352 cut with SmaI, to generate plasmid YEp352/YAP1.
Drug Resistance Assays-For microtiter plate assays, cells grown for 48 h on selective SD Ϫura were resuspended in a saline solution (0.85%) to an A 600 of 0.1. These cells were then diluted 100-fold in SD Ϫura. The diluted cell suspensions were added to round-bottom 96-well microtiter plates (50 l/well; in duplicate) in wells containing equal volumes (50 l) of medium with different concentrations of the drug tested or in drug-free medium. The plates were incubated at 30°C for 48 h. Cell growth was evaluated by reading the optical density at 650 nm in a microplate reader (Vmax™, Molecular Devices). The relative growth was calculated as the percentage of growth in drug-containing medium relative to the control growth in drug-free medium. The chosen concentrations were 50 g/ml for FCZ, 0.1 g/ml for CYH, and 1 M for 4-NQO, which correspond to the minimal inhibitory concentration of these drugs for wild-type strain MRY13-1A transformed with control plasmid YEp352. For the cadmium resistance spot assay, transformants were grown overnight in SD Ϫura medium and approximately 10 4 cells were spotted onto a YPD plate containing 0 or 10 M cadmium. The plates were incubated for 3 days at 30°C. Resistance to hydrogen peroxide was tested essentially as described (35). Briefly, transformants were grown to saturation in SD Ϫura medium and approximately 10 6 cells were spread on YPD plates. A filter paper disc was placed in the center of each plate, 10 l of hydrogen peroxide (30% w/v; Aldrich) was added to the disc and the plates were incubated for 2 days at 30°C before measuring the diameter of the zones of growth inhibition.
RNA Preparation and Northern Blot Analyses-W303 cells transformed with plasmids YEp352, YEp352/CAP1, or YEp352/CAP1-TR were grown in SD Ϫura medium to an A 600 of 2.0. Total RNA was extracted using the glass bead extraction method (59). RNA samples (20 g) were electrophoresed on a 7.5% formaldehyde, 1% agarose gel and transferred by capillarity to a Zeta-Probe nylon membrane (Bio-Rad, Mississauga, Ontario). Detection of specific RNAs was performed by hybridization at 65°C in 0.5 M NaPO 4 , pH 7.2, 1 mM EDTA, 7% SDS, 1% bovine serum albumin, and 100 g/ml salmon sperm DNA with 32 Plabeled DNA probes (60). The CAP1 probe was a 614-bp XbaI-HincII DNA fragment (positions ϩ47 to ϩ661) isolated from clone pF1a. The PDR5 probe was generated by PCR with primers 5Ј-CATACA-GAAGCTCGAATC and 5Ј-CCACAGTTGACTGATAGG and overlaps region ϩ111 to ϩ447 of the PDR5 gene (positions are relative to the translation initiation codon) (10). The YBR008c probe, which overlaps a region from Ϫ2 to ϩ579 of the YBR008c ORF (GenBank Z35877), was amplified by PCR with primers 5Ј-TGGTATACACTTCAACGT and 5Ј-TGTTATGCACAGTAGCAC. A LEU2 probe, consisting of a 0.6-kb ClaI-EcoRV fragment isolated from plasmid pJJ282, was also used as an internal control to monitor RNA loading and transfer (61). Membranes were washed twice at 65°C with a solution containing 40 mM NaPO 4 , pH 7.2, 5% SDS, 1 mM EDTA, 0.5% bovine serum albumin and twice with a solution containing 40 mM NaPO 4 pH 7.2, 5% SDS, and 1 mM EDTA) and were exposed for 24 h at Ϫ80°C.
␤-Galactosidase Assays-An FLR1(YBR008c)-lacZ fusion plasmid was constructed using a PCR fragment overlapping the promoter region, the translation initiation codon, as well as a short portion of the coding region of the FLR1 gene (positions Ϫ828 to ϩ25). This PCR fragment was generated using oligonucleotides 5Ј-CGGGATCCGGTA-GAAGAGTTACGGAA and 5Ј-CCAAGCTTTGTCTGTACGTTGAAGT-GTA, which introduce, respectively, a 5Ј BamHI and a 3Ј HindIII site for directional cloning into YEp368 cleaved with BamHI and HindIII (62), generating plasmid YEp368/FLR1. DNA sequencing was performed to confirm that the FLR1 coding region was in frame with the lacZ gene. MRY13-1A cells carrying plasmid YEp352, YEp352/YAP1, YEp352/CAP1, or YEp352/CAP1-TR were transformed with YEp368/ FLR1. Cotransformants were grown in SD Ϫura Ϫleu medium to an A 600 of 1.0. Cells were collected by centrifugation, rinsed with water, and resuspended in 0.5 ml of Z buffer (60 mM Na 2 HPO 4 , 40 mM NaH 2 PO 4 , 10 mM KCl, 1 mM MgSO 4 , 50 mM ␤-mercaptoethanol) (63). Cell disruption was achieved by vortexing the resuspended pellets for 2 min in the presence of glass beads. After addition of 0.5 ml of Z buffer, the cellular extracts were centrifuged for 15 min. ␤-Galactosidase activities were assayed by measuring o-nitrophenyl-␤-D-galactoside hydrolysis in the supernatants (63) and are expressed as nanomoles of o-nitrophenol formed/min/mg of protein. Protein concentrations were determined by the method of Bradford (64), using bovine serum albumin as standard.

Isolation and Characterization of the C. albicans CAP1
Gene-Molecular genetic techniques involving the isolation of C. albicans genes by functional overexpression in S. cerevisiae provide a powerful approach for the study of drug resistance mechanisms. We have used this approach to search for C. albicans genes that mediate resistance to the azole derivative FCZ when overexpressed in S. cerevisiae. Strain W303 was transformed with a YEp352-based C. albicans genomic DNA library (56). Ura ϩ transformants were pooled and plated on agar plates containing FCZ (500 g/ml), a concentration that corresponds to 10 ϫ the minimal inhibitory concentration for W303 cells transformed with vector YEp352 alone. Plasmids from 12 randomly chosen resistant colonies were purified and analyzed by restriction mapping, revealing that all the isolated plasmids carried an identical 6-kb genomic insert. One of these plasmids (pF1) was selected for further characterization ( Fig.  1). Functional testing of different pF1 subclones indicated that a 3-kb fragment (pF1a) was sufficient to confer resistance ( Fig.  1). DNA sequence analysis of pF1a revealed the presence of an incomplete ORF coding for a C-terminal truncated protein. To obtain a full-length clone for this ORF, a C. albicans fosmid library was screened with a pF1a subfragment as a probe, yielding five positive fosmids. A 3.2-kb BglII fosmid fragment hybridizing with the probe was further subcloned into plasmid YEp352 (57) at the BamHI site (Fig. 1). The resulting plasmid (FosBgl) was also functional in conferring FCZ resistance, as determined by agar plate assay (not shown). DNA sequence analysis of the 3.2-kb BglII genomic DNA fragment revealed that it contains an ORF of 499 amino acids ( Figs. 1 and 2). This protein is rather hydrophilic, with an isoelectric point of 5.15 and a predicted molecular mass of 55 kDa.
A computer search of sequence data bases revealed that the closest homology to the cloned ORF was observed with the S. cerevisiae transcription factor yAP-1 (37% identity, 55% overall homology) (25). The gene was thus named CAP1, for C. albicans AP-1. Other yeast proteins of the AP-1 family found to share a high degree of sequence similarity with the Cap1 protein are S. cerevisiae yAP-2/Cad1 (30% identity, 49% homology) (33,40) and Schizosaccharomyces pombe pap1, which confers resistance to staurosporine (27% identity, 43% homology) (65). The general structure of these proteins includes an N-terminal basic domain involved in DNA binding to target sequences, followed by a leucine zipper motif implicated in dimerization, and a C-terminal acidic domain with regulatory function (25,33,37,40,65,66).
A multiple sequence alignment of Cap1 with its homologues was generated using the Pileup program (58); the two regions displaying the highest degree of sequence conservation are presented in Fig. 3. These regions correspond to the N-terminal basic domain followed by the leucine zipper motif (Fig. 3A) and to the C-terminal acidic domain (Fig. 3B). Within the AP-1 family of transcription factors, Cap1 belongs to a specific subgroup that includes yAP-1, yAP-2, and pap1, according to three criteria. First, its basic domain is located at the N terminus, unlike Gcn4, Fos, or Jun in which the basic domain is located at the C terminus (67). The basic domain contains a short segment highly conserved among the four proteins, which overlaps two clusters of basic residues separated by an alanine spacer motif (Fig. 3A). This specific cluster-spacer-cluster organization, found in all bZip transcription factors, is required for specific DNA binding activity (67). Second, the third position of the heptad repeat of the leucine zipper motif, which is a leucine FIG. 1. Restriction map of a C. albicans genomic DNA clone conferring resistance to FCZ (pF1, top). The thick black lines represent the vector YEp352 (57), and the thin lines represent the genomic DNA insert. The ability of five pF1 subclones (pF1a, -b, -c, -a1, and -a2) to confer FCZ resistance is indicated (ϩ, confers resistance; Ϫ, does not confer resistance). The restriction sites used to generate the different subclones are shown in pF1. The arrow on the pF1 map indicates the position of the initiator methionine at the beginning of the incomplete ORF identified in pF1a. A 3.2-kb BglII fosmid fragment overlapping the complete ORF was subcloned into YEp352 at the BamHI site (FosBgl, bottom). The initiator methionine (arrow) and stop codon (ଙ) are shown in FosBgl.
in Gcn4, Fos, and Jun, differs in all four proteins (an asparagine in both yAP-1 and yAP-2, a threonine in pap1, and an alanine in Cap1) (Fig. 3A). This residue appears to be functionally important as replacement of the asparagine residue with a leucine in yAP-1 (N106L) has been shown to have a strong negative effect on the DNA binding and transactivating activities of this protein (66). Third, the C-terminal acidic region, less conserved than the basic domain, is exclusive to this yAP-1 subfamily of transcription factors. Interestingly, this region contains three cysteine residues invariably conserved among the four proteins (Fig. 3B). In yAP-1, these cysteines are part of three repeated cysteine-serine-glutamate (CSE) sequence motifs recently shown to be required for normal regulation of yAP-1 in mediating oxidative stress response (OSR) (37). Given that the truncated Cap1 protein that lacks the C-terminal region is functional in conferring FCZ resistance (Fig. 1), this domain appears to be dispensable, at least for this specific activity of Cap1.
Functional Complementation of yap1 by CAP1-yAP-1 has been shown to mediate cellular resistance to a large number of toxic compounds, including cadmium and hydrogen peroxide (33)(34)(35). To determine if the structural homology observed between Cap1 and yAP-1 translates into functional homology, we expressed YAP1 and CAP1 in DJY125 cells, which carry a yap1 disruption (35), and compared the ability of these cells to grow in the presence of toxic concentrations of cadmium or hydrogen peroxide. To this end, DJY125 cells were transformed with plasmid YEp352/YAP1 (carrying the entire YAP1 gene under the control of its promoter), YEp352/CAP1 (full-length), YEp352/CAP1-TR (truncated), or YEp352 alone. The S150-2B parental wild-type strain (35) transformed with YEp352 was also included as a control. These transformants were then analyzed by a cadmium resistance spot assay (Fig. 4) and by a hydrogen peroxide filter disc assay (Table I). When compared with S150-2B (YEp352), DJY125 (YEp352) displayed hypersusceptibility to both cadmium and hydrogen peroxide, as reported previously for a yap1 mutant (33,35). As anticipated, overexpression of YAP1 in DJY125 was able to restore the ability of the cells to grow in the presence of cadmium (Fig. 4) or hydrogen peroxide ( Table I). Overexpression of CAP1 in DJY125 was also able to restore the ability of the cells to grow in the presence of both compounds, although with a lower efficiency than YAP1 (Fig. 4 and Table I). Finally, DJY125 cells transformed with YEp352/CAP1-TR were as susceptible to the two compounds as DJY125 cells carrying the vector alone ( Fig. 4 and Table I). These results demonstrate that Cap1 and yAP-1 are functionally related and that the C-terminal domain of Cap1 is required to mediate resistance to cadmium and hydrogen peroxide in S. cerevisiae DJY125 cells.
Identification of the YBR008c ORF as a Potential Transcriptional Target of CAP1-It has been shown that YAP1 confers resistance to toxic compounds and to oxidative stress by transcriptionally activating a number of downstream target genes encoding effector proteins mediating the resistance (34,36,38,39). It is thus likely that Cap1 mediates FCZ resistance in S. cerevisiae through transcriptional activation of similar effector genes. What could these effectors be? In C. albicans, overexpression of CDR1, CDR2, and BEN r has been shown to be associated with FCZ resistance (47). This raised the possibility that homologues of these genes could be the CAP1 target(s) mediating FCZ resistance in S. cerevisiae.
To test this hypothesis, we generated gene-specific probes for PDR5, the closest S. cerevisiae homologue of CDR1 and CDR2 (10), and for YBR008c, the closest BEN r homologue in S. cerevisiae identified through a Blast search. As for BEN r , the YBR008c gene product is predicted to be a member of the MF superfamily (5,7,68). Total RNA was extracted from S. cerevisiae W303 cells transformed with plasmids YEp352, YEp352/ CAP1, or YEp352/CAP1-TR. RNA samples were analyzed by Northern blotting using a CAP1 (Fig. 5A), a PDR5 (Fig. 5B), or a YBR008c (Fig. 5C) radiolabeled DNA fragment as probe. The membranes were hybridized concurrently with a LEU2 probe as a control (61). RNA analysis with the CAP1 probe showed that the full-length CAP1 gene was expressed at high levels in S. cerevisiae, giving rise to two RNA transcripts of 2.2 and 1.9 kb (Fig. 5A, lane 2), which were absent from the W303 control FIG. 2. Nucleotide and deduced amino acid sequence of the CAP1 gene. Standard and bold numbers (left) refer to the nucleotide and to the amino acid sequences, respectively. The two boxed regions in the coding sequence represent domains that are highly conserved between the predicted Cap1 protein and its homologues (see Fig. 3). The N-terminal box defines a basic region followed by a leucine zipper motif (residues defining this motif are boxed), and the C-terminal box delineates an acidic region (25). The position of the truncation in the incomplete pF1 clone is indicated by an arrow at position 1001. cells (Fig. 5A, lane 1). Multiple transcripts were observed in cells expressing the truncated CAP1 gene (Fig. 5A, lane 3), probably resulting from read-through transcription within the plasmid due to the lack of proper transcription termination signals in the truncated gene. The basal level of PDR5 expression was found to be slightly induced in cells transformed with CAP1, either full-length or truncated (Fig. 5B). Finally, YBR008c expression, undetectable in control cells (Fig. 5C,  lane 1), was dramatically induced in W303 cells overexpressing either the full-length or the truncated CAP1 gene (Fig. 5C,  lanes 2 and 3). The massive overexpression of YBR008c in these cells is similar to that observed for BEN r in some FCZ resistant clinical and experimental isolates (47,48). These results are consistent with the hypotheses that YBR008c is a target gene for Cap1 in this heterologous expression system and is potentially the Cap1 effector mediating FCZ resistance in S. cerevisiae CAP1 transformants.

YBR008c Is Essential for CAP1-and YAP1-mediated FCZ
Resistance-To determine whether YBR008c was this effector, we constructed a yeast strain carrying a chromosomal deletion of this gene. To this end, a chimeric gene was generated in which a 1.6-kb SpeI-HindIII fragment overlapping the ATG initiator codon and most of the coding region of YBR008c was replaced with the HIS3 gene, and was introduced into the isogenic diploid strain W303. A selected His ϩ transformant carrying one wild-type YBR008c and one disrupted ybr008c⌬::HIS3 allele (as confirmed by Southern blotting analysis; data not shown) was sporulated. Tetrad dissection gave rise to viable ybr008c⌬::HIS3 spores, demonstrating that YBR008c is not essential for growth. Two sister spores, MRY13-1A (YBR008c) and MRY13-1B (ybr008c⌬::HIS3), were selected for the following resistance studies.
Strains MRY13-1A and MRY13-1B were transformed with plasmids YEp352, YEp352/YAP1, YEp352/CAP1, and YEp352/ CAP1-TR. These transformants were tested for their ability to grow in the presence or absence of 50 g/ml FCZ (the minimal inhibitory concentration for wild-type strain MRY13-1A transformed with control plasmid YEp352), using a microtiter plate assay (Fig. 6A). We found that the three genes (YAP1, CAP1, and CAP1-TR) were able to confer FCZ resistance when expressed in the wild-type YBR008c strain, demonstrating that the ability of CAP1 to mediate FCZ resistance in S. cerevisiae is not restricted to the C. albicans gene but also extends to its S. cerevisiae homologue. This finding further expands the spectrum of drugs to which YAP1 has been shown to confer resistance. Interestingly, the full-length Cap1 protein was approximately 3 times less active than the truncated Cap1 protein in this assay, indicating that the C-terminal domain of Cap1 behaves as an inhibitor of Cap1 activity with respect to the transcriptional regulation of YBR008c in S. cerevisiae (Fig. 6A). Unlike MRY13-1A cells however, MRY13-1B cells transformed with plasmids YEp352/YAP1, YEp352/CAP1, or YEp352/CAP1-TR were completely unable to grow in FCZ, exhibiting a relative growth value identical to that of the YEp352 control transformants (Fig. 6A). These results demon-  (35) transformed with plasmid YEp352, YEp352/YAP1, YEp352/CAP1, or YEp352/CAP1-TR and S150-2B cells (35) transformed with YEp352 were tested for resistance to cadmium by a spot test growth inhibition assay. Transformants were grown overnight in SD Ϫura medium. Approximately 10 4 cells were spotted onto a YPD plate containing 10 M cadmium (ϩ CdSO 4 ) or no cadmium (Ϫ CdSO 4 ). The plates were incubated for 3 days at 30°C. strate that YBR008c is essential for YAP1-and CAP1-mediated FCZ resistance. Since MRY13-1B cells transformed with plasmids YEp352/YAP1, YEp352/CAP1, or YEp352/CAP1-TR were no more resistant to FCZ than the YEp352 control transformants, we conclude that no other YAP1 or CAP1 downstream targets, besides YBR008c, contribute to the FCZ resistance phenotype observed in the corresponding MRY13-1A transformants. In particular, these results rule out a potential contribution of the PDR5 gene suggested by the slightly elevated levels of PDR5 RNA transcripts observed in the W303 YEp352/ CAP1 and YEp352/CAP1-TR transformants (Fig. 5B). Finally, MRY13-1B (YEp352) cells were not more susceptible to FCZ than MRY13-1A (YEp352) cells, as predicted from the lack of YBR008c constitutive expression in the absence of CAP1 overexpression (Fig. 5C). Taken together, these data demonstrate that YBR008c codes for the YAP1 and CAP1 effector protein conferring FCZ resistance in S. cerevisiae. YBR008c was thus renamed FLR1, for fluconazole resistance 1.
YAP1-and CAP1-mediated FLR1 Overexpression Confers MDR-Overexpression of YAP1 in S. cerevisiae has been shown to confer resistance to the toxic compounds CYH and 4-NQO, but the target(s) of yAP-1 mediating this resistance has not yet been identified (31,69). Interestingly, it has been shown that overexpression of the BEN r gene in S. cerevisiae confers resistance to both CYH and 4-NQO and that inactivation of BEN r in C. albicans results in hypersusceptibility to these two compounds (47,53,70). It was thus of interest to investigate if FLR1 was the YAP1 effector mediating CYH and 4-NQO resistance in S. cerevisiae.
To this end, strains MRY13-1A and MRY13-1B transformed with plasmids YEp352, YEp352/YAP1, YEp352/CAP1, and YEp352/CAP1-TR were examined for their ability to grow in the presence of CYH or 4-NQO (Fig. 6, B and C). We found that overexpression of the three genes (YAP1, CAP1, and CAP1-TR) in the wild-type FLR1 strain resulted in resistance to both CYH and 4-NQO (Fig. 6, B and 6C). As observed for FCZ (Fig.  6A), the full-length CAP1 gene was much less active than the truncated CAP1 in conferring resistance to these two compounds (Fig. 6, B and C). MRY13-1B cells transformed with plasmids YEp352/YAP1, YEp352/CAP1, or YEp352/CAP1-TR were as susceptible to CYH as the YEp352 control transformants (Fig. 6B), indicating that FLR1 is the only YAP1 target mediating CYH resistance in S. cerevisiae. In contrast to FCZ and CYH, however, MRY13-1B cells transformed with   (35) transformed with plasmid YEp352, YEp352/YAP1, YEp352/CAP1, or YEp352/CAP1-TR and S150 -2B cells (35) transformed with YEp352 were tested for their level of resistance to hydrogen peroxide. Strains were grown to saturation in SD Ϫura medium, and approximately 1 ϫ 10 6 cells were plated on YPD plates. A filter paper disc was placed in the center of each plate, 10 l of H 2 O 2 (30%) was added to the disc, and the plates were incubated for 2 days at 30°C. The diameters of the clear zones were measured (cm). The data shown represent the means of three independent experiments performed in triplicate.

Strains
Diameter of zone of growth inhibition YEp352/YAP1 retained a significant level of residual resistance to 4-NQO (Fig. 6C), revealing the participation of other YAP1controlled molecular determinant(s) of 4-NQO resistance in S. cerevisiae besides FLR1. Although almost as active as YAP1 in conferring resistance to 4-NQO in FLR1 wild-type cells, the truncated CAP1 gene was unable to induce the expression of these other 4-NQO resistance determinants, as no residual resistance was observed in MRY13-1B (YEp352/CAP1-TR) as compared with MRY13-1B (YEp352/YAP1) transformants (Fig. 6C). Taken together, these results demonstrate that FLR1 is the only YAP1 target mediating resistance to FCZ and CYH and is one of the YAP1 targets conferring resistance to 4-NQO. Thus, we can conclude that FLR1 represents a new YAP1controlled multidrug resistance molecular determinant in S. cerevisiae. FLR1 Is a Transcriptional Target for Proteins of the yAP-1 Family-To determine whether FLR1 is under the transcriptional control of the yAP-1 and Cap1 proteins, we assessed their ability to activate transcription of a lacZ reporter gene under the control of the FLR1 promoter. To this end, an FLR1-lacZ fusion gene was constructed, consisting of 828 bp of 5Ј noncoding sequence and 25 bp from the FLR1 gene fused to E. coli lacZ in plasmid YEp368 (62). This FLR1-lacZ gene fusion was then introduced into MRY13-1A cells carrying plasmids YEp352/YAP1, YEp352/CAP1, YEp352/CAP1-TR, or YEp352. The ␤-galactosidase activities were determined from the resulting cotransformants to measure the effect of these plasmids on the expression of the FLR1-lacZ fusion gene (Fig. 7). Presence of the FLR1-lacZ construct in cells carrying the control plasmid YEp352 gave rise to 0.05 units of ␤-galactosidase activity, a small but significant value since use of a control YEp368 vector lacking the FLR1 promoter did not result in any measurable ␤-galactosidase activity in the same assay (data not shown). When the FLR1-lacZ plasmid was introduced in cells transformed with plasmid YEp352/YAP1, a ␤-galactosidase activity of 5.2 units was obtained, representing an approximate 100fold increase as compared with the value measured in the control YEp352 transformants. This result is consistent with the proposition that FLR1 is under the transcriptional control of YAP1 in S. cerevisiae. A similar level of ␤-galactosidase activity was obtained with cells transformed with the YEp352/ CAP1 plasmid (5.7 units), confirming the functional relatedness between yAP-1 and Cap1. Finally, the ␤-galactosidase activity measured in cells transformed with the YEp352/ CAP1-TR was approximately 2-fold lower than that measured in cells transformed with YEp352/CAP1 (3.0 units), indicating that the truncated Cap1 protein is less active than the fulllength Cap1 protein in this transactivation assay. These results were unexpected, in light of our data showing that the truncated Cap1 protein was more active than full-length Cap1 in conferring FCZ, CYH, and 4-NQO resistance (Fig. 6). Nevertheless, these data show that both Yap1 and Cap1 are capable of stimulating FLR1 transcription and provide evidence that this control is mediated through the FLR1 promoter.
These results prompted us to scan the promoter region of the FLR1 gene for the presence of a DNA sequence motif similar to previously identified YREs (34). Our analysis revealed that the FLR1 promoter indeed contains three potential YREs of sequence 5Ј-TTAGTA/CA, one on the coding strand (position Ϫ149 with respect to the translation initiation codon at ϩ1) and two on the anticoding strand (positions Ϫ161 and Ϫ358) (Fig. 8). Functional YREs of similar sequence have been shown to be present in the promoter of the YAP1 target genes GSH1, YCF1, TRX2, and GLR1 (Fig. 8) as well as in the SV40 early enhancer element (34,36,38,39,71). A detailed functional analysis of the FLR1 promoter is currently under way to determine whether the control of FLR1 transcription by yAP-1 is direct or indirect and whether it is mediated through the YREs present in the promoter. DISCUSSION Investigation of C. albicans molecular determinants of FCZ resistance has allowed us to isolate a gene coding for a new member of the yeast AP-1 family that we have named CAP1. A computer search of protein data bases identified S. cerevisiae yAP-1 as being the closest homologue of the Cap1 protein (25). Other yeast proteins also found to share a high degree of sequence similarity with Cap1 are S. cerevisiae yAP-2/Cad1 and S. pombe pap1 (33,40,65). These proteins are apparently not only structurally but also functionally related, since their overexpression from a multicopy plasmid results in a similar phenotype, namely an increased cellular resistance to different toxicants (31,33,40,65,69). More specifically, we show that the sequence homology between the Cap1 and yAP-1 proteins translates into functional homology, because (a) CAP1 expression in a yap1 mutant partially restores the ability of the cells to grow on otherwise toxic concentrations of cadmium or hydrogen peroxide (Fig. 4 and Table I) and (b) both yAP-1 and Cap1 can transactivate FLR1 to confer MDR (Figs. 6 and 8). yAP-1 is the best studied member of this family of fungal transactivators. In addition to its role in drug resistance, yAP-1 has been shown to participate in OSR via the transcriptional activation of a spectrum of targets including GSH1, encoding ␥-glutamylcysteine synthase (38); TRX2, one of the two genes coding for thioredoxin (36); and GLR1, encoding glutathione reductase (39). Therefore, it is quite possible that Cap1 serves similar functions in C. albicans by transactivating a number of effector genes homologous to those controlled by yAP-1 in S. cerevisiae. The availability of the cloned CAP1 gene will now allow an analysis of its role in C. albicans with respect to both MDR and OSR.
An amino acid sequence comparison of the Cap1, yAP-1, yAP-2, and pap1 proteins indicates that the most conserved regions among these proteins overlap the N-terminal bZip and the C-terminal acidic domains (Fig. 3). A bZip domain is found in a large number of transcription factors of the AP-1 family, including S. cerevisiae Gcn4 and mammalian Jun, Fos, C/EBP, and CREB, and its function in DNA binding and in protein dimerization has been extensively studied (67). However, the role of the C-terminal domain, which is present only in the yeast AP-1 subfamily of transcription factors, is much less understood. A short segment of approximately 60 amino acids located at the extreme C terminus contains a number of highly conserved residues, including three invariably conserved cysteines (33,40,65) (Fig. 3B). In yAP-1, these cysteines are part of three repeated cysteine-serine-glutamate (CSE) sequence motifs recently shown to be required for normal regulation by yAP-1 in mediating oxidative stress tolerance (37). It has been shown that truncated yAP-1, yAP-2, and pap1 proteins lacking the C-terminal domain are still functional in conferring resistance to cadmium, to 1,10-phenanthroline, and to staurosporine, respectively, indicating that this domain is dispensable at least for these specific phenotypes (40,65,66). During the course of our work, we have isolated a genomic DNA fragment carrying an incomplete version of the CAP1 gene coding for a truncated protein lacking the entire C-terminal domain (Fig.  1). On the one hand, we find that the truncated Cap1 protein is active in conferring FLR1-mediated resistance to FCZ, CYH, and 4-NQO upon overexpression in a wild-type strain (Fig. 6). On the other hand, when tested for its ability to suppress a yap1 mutation, the truncated Cap1 protein is completely inactive in restoring tolerance to cadmium and hydrogen peroxide ( Fig. 4 and Table I). A potential explanation for these two different phenotypes is that the truncated Cap1 protein requires the presence of yAP-1 to bring about efficient transcriptional activation. Alternatively, it is possible that the C-terminal domain of Cap1 selectively modulates the protein activity, displaying either an inhibitory or an activating function depending on the transcriptional target. The fact that the truncated Cap1 protein (a) is active in inducing FLR1-dependent 4-NQO resistance but inactive in inducing FLR1-independent 4-NQO resistance (Fig. 6C) and (b) is able to confer FCZ resistance in yap1 DJY125 cells (data not shown) supports the second hypothesis. Finally, we find that the truncated Cap1 protein, which is more active than the full-length protein in conferring MDR, is less active than full-length Cap1 in transactivating an FLR1-lacZ fusion gene (compare Figs. 6 and 7). One possible reason for this discrepancy may be that drugs, present in the drug resistance assay and absent from the transactivation assay, differentially modulate the activity of the two forms of Cap1. As C. albicans genes can behave as mutants when expressed in S. cerevisiae (72), it will be important to study the effect of the Cap1 C-terminal truncation in C. albicans and/or to test the effects of a similar deletion in yAP-1 on its ability to transactivate its different targets in S. cerevisiae to be able to draw definitive conclusions from these observations.
Several ORFs predicted to code for MDR transporters of the MF superfamily (MFS) based on structural considerations have been identified during the yeast genome sequencing project but their role in MDR remains to be determined (5,7). Based on hydropathy analyses, these ORFs have been grouped into three clusters: members of cluster I (12 ORFs) are predicted to contain 12 TM domains, whereas members of cluster II (10 ORFs) and of cluster III (6 ORFs) are predicted to contain 14 TM domains (5,7). So far, only two transporters of the MFS in S. cerevisiae have been demonstrated to function as MDR determinants, namely Atr1 (4-NQO and aminotriazole resistance) and Sge1 (crystal violet and ethidium bromide resistance), which both belong to cluster II (reviewed in Ref. 7). Our data, showing that YAP1-mediated FLR1 overexpression in S. cerevisiae results in cellular resistance to FCZ, CYH, and 4-NQO, demonstrates that FLR1 indeed functions as an MDR determinant (Fig. 6). Furthermore, overexpression of FLR1 from the strong constitutive glyceraldehyde-3-phosphate dehydrogenase promoter in S. cerevisiae results in high levels of resistance to these three compounds as well as to benomyl and 1,10-phenanthroline, confirming that FLR1 is an MDR determinant with broad substrate specificity. 2 FLR1 (YBR008c) is predicted to code for an integral membrane protein with 12 potential transmembrane segments belonging to cluster I (5, 7). Interestingly, MFS proteins known to function as MDR efflux pumps in other yeasts also belong to cluster I, including C. albicans BEN r , Candida maltosa CYHR, and S. pombe CAR1 (7). It will be of interest to determine whether other MFS MDR ORFs, identified based on structural considerations by the systematic analysis of the yeast genome, actually function as MDR determinants and whether some of them are under the transcriptional control of YAP1 as part of a yet unidentified MDR network in S. cerevisiae (see below).
A number of observations indicate that FLR1 expression is under the control of AP-1 proteins. First, the steady state level of FLR1 RNA is drastically increased in CAP1 and CAP1-TR transformants (Fig. 5C). Second, YAP1-and CAP1-mediated multidrug resistance in a wild-type strain is completely abrogated (FCZ and CYH) or strongly reduced (4-NQO) in an flr1 null mutant strain (Fig. 6). Third, the FLR1 promoter contains three DNA sequence elements matching a functional YRE (Fig.  8). Fourth, the FLR1 promoter fused to a reporter gene is strongly transactivated by the overexpression of YAP1 or CAP1 (Fig. 7). Overexpression of YAP1 in S. cerevisiae had been previously shown to confer resistance to different toxic compounds including CYH and 4-NQO, but the target(s) of yAP-1 mediating this resistance had so far remained unidentified (31,69). The results presented here clearly identify FLR1 as being the YAP1 effector mediating CYH resistance (in addition to FCZ resistance) and one of the YAP1 effectors conferring resistance to 4-NQO. Taken collectively, these results allow us to conclude that FLR1 represents a new YAP1-controlled MDR molecular determinant in S. cerevisiae. This widens the spectrum of already known yAP-1 physiological targets to include an MFS MDR transporter and further substantiates that yAP-1 is a key regulator molecule involved in controlling MDR in addition to OSR. It also raises the possibility that yAP-1 regulates additional unidentified targets involved in MDR. Our results showing residual YAP1-mediated resistance to 4-NQO in an flr1 null mutant strain confirms this hypothesis (Fig. 6C). Such YAP-1 target(s) mediating 4-NQO resistance could well include the ABC transporter SNQ2 and/or the major facilitator ATR1, as both genes have been shown to confer resistance to 4-NQO and to contain YRE motifs in their promoter regions (14,20,21,73). Experiments are currently under way to test this proposition.
C. albicans strains can develop clinical resistance to FCZ in individuals undergoing long term therapy or prophylactic treatment, mostly AIDS patients (42)(43)(44). Two major mechanisms of FCZ resistance have been identified so far in these strains: (a) alterations in the drug target (14-␣-sterol demethylase, the product of the ERG16 gene), resulting in an increased production of the enzyme or in its reduced binding affinity for FCZ (reviewed in Refs. 74 and 75); and (b) a reduced intracellular FCZ accumulation, which usually correlates with the overexpression of the ABC transporter genes CDR1 and CDR2 and of the MF transporter BEN r (47,51); this latter mechanism appears to be the most important. The molecular events upregulating the expression of these transporters in C. albicans are not known. It has been shown by Northern blot analyses of C. albicans clinical isolates that CDR1 is constitutively expressed at low levels in FCZ-susceptible strains, while CDR2 and BEN r are not expressed in these strains, at least at levels detectable by this technique (47,51). However, these genes become markedly overexpressed in FCZ-resistant isolates, which can exhibit either CDR1, concomitant CDR1 and CDR2, or BEN r overexpression (47,48,51), suggesting the participation of transporter-specific transactivating factors in the establishment of FCZ resistance in these isolates. In S. cerevisiae, the expression of PDR5, a close homologue of CDR1 and CDR2, is under the transcriptional control of the zinc finger proteins PDR1 and PDR3 (27); it is thus possible that PDR1 and PDR3 homologues in C. albicans also operate to up-regulate CDR1 and CDR2 expression. On the other hand, our isolation of a YAP1 homologue from C. albicans together with our demonstration that both yAP-1 and Cap1 transcriptionally control the BEN r homologue FLR1 in S. cerevisiae suggest that CAP1 could be involved in controlling BEN r expression to cause FCZ resistance in C. albicans. We have found that overexpressing CAP1 (the truncated form) in C. albicans CAI4 cells results in BEN r overexpression and in FCZ resistance, confirming that indeed CAP1 participates in the transcriptional control of BEN r in C. albicans. 3 What could be the molecular events leading to BEN r transcriptional activation? FCZ resistance in strains overexpressing BEN r is a stable phenotype, suggesting that FCZ resistance in these strains is due to genetic alterations rather than to transient cellular adaptations (47,48). Such permanent alterations potentially include dominant activating mutations in the BEN r promoter or in trans-acting factors controlling BEN r expression, such as CAP1. We are currently investigating these possibilities in clinical and experimental FCZ resistant C. albicans isolates overexpressing BEN r .


extfieldsofstudy: Biology Medicine

text:
Allosteric Regulation of RecA Protein Function Is Mediated by Gln194 *

Binding of ATP to the RecA protein induces a high affinity DNA binding required for activation of enzyme function. Screens for in vivo recombination and repressor cleavage activities show Gln194 to be intolerant of all substitutions. Analyses of three mutant proteins (Q194N, Q194E, and Q194A) show that although basal enzyme function is maintained, each protein no longer displays an ATP-induced increase in DNA binding affinity. High salt activation of RecA function is also disrupted by these mutations. In contrast, ATP-induced changes in the oligomeric structure of RecA are maintained in the mutant proteins. These results demonstrate that Gln194 is a critical “allosteric switch” for ATP-induced activation of RecA function but is not the exclusive mediator of ATP-induced changes in RecA.

The Escherichia coli RecA protein is a multifunctional enzyme that plays a central role in the processes of recombinational DNA repair, homologous genetic recombination, and the cellular SOS response to DNA damage (1)(2)(3). Each of these activities exhibits a common initial or activating step, formation of a RecA-ATP-ssDNA 1 nucleoprotein filament (4 -6). The binding of RecA to ssDNA is regulated in a classic allosteric fashion, whereby the binding of ATP induces a high affinity DNA binding state of the protein (7,8). In the presence of ADP, or in the absence of cofactor, RecA exhibits a low affinity (Ͼ20 M) for ssDNA (7).
The crystal structure of the helical RecA protein filament has been solved in both the absence and presence of ADP (9, 10) and displays a helical pitch (82.7 Å) which is intermediate between the inactive nucleotide-free form (Ϸ70 Å) and the active ATPbound form (Ϸ95 Å) as determined by electron microscopy (reviewed in Ref. 11). Despite this, the ATP binding site shows a remarkable conservation of structure compared with several other nucleotide-binding proteins (e.g. p21 ras , EF-Tu, and adenylate kinase), and Story and Steitz (10) were able to model specific determinants of both ATP binding and hydrolysis in the RecA structure. In addition, the structure provided valuable insight into a possible allosteric mechanism for ATP-induced high affinity binding to ssDNA. The carboxyamide side chain of Gln 194 extends into the ATP binding site and would be in very close proximity to the ␥-phosphate of bound ATP (Fig.  1). Gln 194 immediately precedes one of two disordered loops in the structure (L2, residues 195-209) that were proposed to be part of the DNA binding sites (9). Recent work has, in fact, provided strong evidence that L2 comprises all or a large part of the ssDNA binding site within RecA (12)(13)(14). Story and Steitz (10) proposed that upon ATP binding Gln 194 interacts with the nucleotide ␥-phosphate thereby causing L2 to assume a conformation with high affinity for ssDNA. Upon ATP hydrolysis this interaction would be lost, returning L2 to a low affinity DNA binding conformation.
In this study, we show that mutations at Gln 194 prohibit the formation of a high affinity ssDNA binding state when ATP is bound. In addition, mutations at Gln 194 block the high salt activation of RecA function. These results indicate that Gln 194 is an important "on-off" switch required for the general activation of RecA function.

EXPERIMENTAL PROCEDURES
Materials-Labeled NTPs and dNTPs were from NEN Life Science Products. PEI-cellulose chromatography plates were from J. T. Baker Inc. Single-stranded RV-1 DNA, an M13 derivative, was used for in vitro ATPase and repressor cleavage assays and was purified as described (15). An 86-base oligonucleotide used in the ssDNA binding assays and mutagenic oligonucleotides were made with an Applied Biosystems 392 DNA/RNA synthesizer. Double-stranded DNA (dsDNA) used in both the ATPase and dsDNA binding assays was NcoI-linearized pTRecA220 (Ϸ5, 500 base pairs (16)). LexA protein was a generous gift from Dr. John Little and Donald Shepley (Dept. of Biochemistry, University of Arizona). Restriction enzymes, T4 DNA ligase, T4 polynucleotide kinase, and Klenow DNA polymerase I large fragment were from New England Biolabs. Sequenase version 2.0 was from U. S. Biochemical Corp. Nitrocellulose filters were from Schleicher and Schuell. Isopropyl-1-thio-␤-D-galactopyranoside and mitomycin C were from Sigma.
Mutagenesis-Mutations were introduced at position 194 using a modification of a previously described cassette mutagenesis procedure (17). Two 81-base oligonucleotides corresponding to the top and bottom strands encoding residues Thr 186 to Asn 213 were synthesized such that codon 194 read NN(G/C) and all other bases were the wild type recA sequence. Oligonucleotides were annealed, and the resulting cassette was ligated into AflIII/MluI-digested pTRecA332, a derivative of pTRecA322 (18) containing a unique AflIII site at position 187 and a unique MluI site at position 214. Plasmids were transformed into a ⌬recA strain, DE1663Ј (18), and colonies were selected on LB-ampicillin plates. Amino acid substitutions were determined by DNA sequence analysis.
Recombinational DNA Repair Activity in Vivo-The recombinational DNA repair activity of each mutant protein was determined using two genetic screens as described previously (18), cell survival in the presence of mitomycin C and cell survival following exposure to different doses of UV light.
Determination of the Size of Mutant RecA Protein Oligomers-The oligomeric distribution of wild type and mutant RecA proteins was determined using gel filtration chromatography as described previously (19).
Purification of Wild Type and Mutant RecA Proteins-Wild type and mutant proteins (Q194A, Q194E, and Q194N) were purified using a previously described method (20) and were determined to be Ͼ95% pure * This work was supported by National Institutes of Health Grant GM44772 (to K. L. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
as judged by Coomassie-stained gels. Proteins were quantitated using the Bio-Rad protein assay kit and by optical density, using an extinction coefficient of ⑀ 280 ϭ 0.59 mg Ϫ1 ⅐ml (21). No exonuclease activity was detected in any of these preparations, even at elevated concentrations of RecA (50 M) in the nuclease assay. None of these mutations has any deleterious effect on the overall folded structure or thermal stability of the protein. Wild type RecA and each mutant protein showed identical circular dichroism profiles and measurements of 222 nm as a function of temperature (5-90°C) gave a T m ϭ 53 Ϯ 1°C for all 4 proteins (data not shown).
RecA-mediated LexA Cleavage-RecA-mediated cleavage of the LexA repressor was measured in vivo using strain DE1663Ј as described previously (18). The ability of purified wild type and mutant RecA proteins to mediate the autocleavage of the LexA repressor in vitro was assessed in the presence of ssDNA as described previously (20) and in the presence of high salt using the following modifications of a previously described method (22). Reaction buffer contained 50 mM K 2 HPO 4 / KH 2 PO 4 , 10 mM Mg(OAc) 2 , 0.6 M NaOAc, 5 mM ATP␥S, 5% glycerol and was brought to a final pH of 7.0. Reactions (60 l) were performed at 37°C and contained RecA protein (0.5 mg/ml) which had been preincubated with 5 mM ATP␥S. LexA protein was added to a final concentration of 1.0 mg/ml. During the time course samples (8 l) were removed and EDTA was added to 100 mM. Intact LexA and cleavage products were resolved on a 15% SDS-polyacrylamide gel, stained with Coomassie Brilliant Blue, and quantitated using scanning densitometry (20).
ATPase Activity-Hydrolysis of ATP was measured under 3 different conditions, in the presence of ssDNA, dsDNA, or 1.8 M NaCl. The ssDNA-dependent ATPase activity was measured as described previously (20,23). The dsDNA-dependent ATPase activity was measured essentially as described (23). Reaction mixtures contained 20 mM sodium maleate (pH 6.2), 10 mM ssDNA Binding-Nitrocellulose filter binding assays were performed using a variation of a described procedure (25) and an apparatus similar to that described by Wong and Lohman (26). Filters were prepared by soaking in 0.4 M KOH for 10 min followed by several washes with double distilled H 2 0 until the pH approximated 7.0. Filters were equilibrated in binding buffer for at least 1 h prior to use. Reaction mixtures (50 l) contained binding buffer (20 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 1 mM DTT, 0.5 mM EDTA, 20 mM NaCl), 40 M 5Ј-end-labeled ssDNA (concentration of bases), and where indicated, 0.5 mM ATP␥S. Reactions were started with the addition of the indicated amounts of protein and incubated at 37°C for 15 min. Samples were applied to the filter under suction, and the filters were washed with 2.0 ml of binding buffer containing 150 mM NaCl. Filters were air-dried, and bound DNA was quantitated by analyzing air-dried filters with a Molecular Dynamics PhosphorImager and ImageQuant software (v5.6).
dsDNA Binding-Nitrocellulose filter binding assays were performed using a variation of a previously described procedure (25). Mixtures were placed on ice, 4 cm from an 8-watt mercury vapor lamp (Sylvania G8T5), and irradiated for 45 min. Crosslinked products were resolved on a 10% SDS-polyacrylamide gel and analyzed using a Molecular Dynamics PhosphorImager and ImageQuant software (v5.6).

RESULTS AND DISCUSSION
RecA Activity in Vivo-We obtained 16 of 19 possible substitutions at Gln 194 , including a conservative Asn, an isosteric Glu, and an Ala. 2 In vivo screens for recombinational DNA repair and LexA cleavage showed that these activities are completely dependent on the wild type Gln residue (data not shown). Each of the 16 mutants showed no survival greater than the ⌬recA control in the presence of mitomycin C or following exposure to UV, even under low dose conditions (0.3 g/ml mitomycin C or 0.67 J/m 2 /s UV light). In addition, the LexA coprotease activity of each mutant was completely inhibited, a result which was confirmed by in vitro LexA cleavage assays (see below).
ssDNA Binding-RecA exhibits two distinct binding affinities for ssDNA, a low affinity in the absence of any nucleotide cofactor or the presence of ADP, and a high affinity in the presence of cofactor (7,8). We show that in the absence of nucleotide wild type RecA protein binds to ssDNA with an apparent K d Ϸ20 M (Fig. 2A). Each mutant protein tested, Q194A, Q194E, and Q194N, exhibits a comparable apparent affinity, although the Q194N protein appears to have a slightly reduced binding capacity ( Fig. 2A). In the presence of ATP␥S, however, a dramatic increase is seen in the affinity of wild type RecA for ssDNA compared with the mutant proteins (Fig. 2B). Wild type RecA now binds the ssDNA substrate with an apparent K d Ϸ1 M, whereas none of the 3 mutant proteins shows any change in affinity for ssDNA. These results indicate that the mutations at position 194 render the proteins unable to undergo the ATP-induced transition required for high affinity ssDNA binding.
ATP Binding-An essential control was to test the ability of the mutant proteins to bind ATP. Using a UV crosslinking procedure we show that ATP binding by each of the 3 mutant proteins (Q194A, Q194E, and Q194N) is very similar to that of wild type RecA (Fig. 3). Proteins that had been heat-denatured prior to UV exposure show no crosslinking. These results show that the inability of the mutant proteins to assume a high affinity ssDNA binding state is not due simply to a defect in ATP binding.
ATP-induced Effects on the Oligomeric Distribution of RecA-In solution RecA protein exists as a heterogeneous population of oligomers, ranging in size from monomers, dimers, and hexamer-sized rings to larger filaments and bundles of filaments (27)(28)(29)(30). In the presence of ATP this distribution is shifted such that bundles of filaments are disrupted and the average filament length is shorter (27)(28)(29). We used a recently developed gel filtration assay (19) to assess any effect that mutations at position 194 may have on the oligomeric properties of RecA, as well as the effect of ATP on the oligomeric distribution. Our results show that substitution of Gln 194 with Ala, Glu, or Asn has no effect on the oligomeric distribution of RecA and importantly, that each mutant protein shows an ATP-induced shift in this distribution identical to that observed for wild type RecA (Fig. 4). Addition of ATP␥S shifts the peak representing long RecA filaments and bundles to a smaller size (Fig. 4 and Ref. 19). These results again show that each mutant RecA protein is capable of binding ATP and that conformational changes associated with the ATP-induced shift in the distribution of RecA oligomers are maintained and therefore not transmitted through Gln 194 .
ATPase Activity-The ATPase activity of RecA protein is stimulated upon binding to either ssDNA or dsDNA (23). In the absence of DNA wild type RecA exhibits a low level ATPase activity at pH 7.5 (V i /E ϭ 0.2) whereas in the presence of a ssDNA cofactor activity increases significantly (V i /E ϭ 17.4; Fig. 5). While the basal rate of ATP turnover for the mutant proteins is similar to that of wild type RecA (V i /E Ϸ0.2; Fig.   5A), no ssDNA-dependent increase in activity is observed (Fig. 5B).
At pH 6.2 the catalytic efficiency of RecA increases approximately 30-fold in the presence of dsDNA (23). Each of the Gln 194 mutant proteins maintains a wild-type level of DNAindependent ATPase activity at pH 6.2 (V i /E ϭ 0.5). However, an increase in the presence of dsDNA was seen only with wild type RecA (data not shown). Control experiments showed that wild type and mutant proteins bound equivalent low levels of dsDNA in the absence of ATP␥S, yet only wild type RecA displayed a significant ATP-dependent increase in dsDNA binding (data not shown).
These results demonstrate that mutations at Gln 194 block the DNA-dependent activation of ATPase activity but do not affect the basal level of DNA-independent ATP turnover.
LexA Coprotease Activity-In the presence of both nucleotide and ssDNA cofactors RecA mediates the autoproteolysis of the LexA repressor (5). While wild type RecA catalyzes a significant level of LexA autoproteolysis (95% cleaved/40 min) this activity is completely lacking in each of the 3 mutant proteins (data not shown).
High Salt Activation of RecA Activities-In the absence of any DNA cofactor, but in presence of high salt, wild type RecA protein is activated for both ATP hydrolysis (24) and LexA cleavage (22). Because mutations at Gln 194 prohibit high affinity DNA binding required for both of these activities we determined whether the Q194A, Q194E, and Q194N mutations also affected high salt activation of RecA function. We found that although the ATPase activity of wild type RecA was greatly stimulated in the presence of 1.8 M NaCl (V i /E ϭ 23) the 3 mutant proteins showed only very low activity under these conditions (Fig. 6). Similarly, in the presence of 0.6 M NaOAc wild type RecA catalyzed a significant level of LexA autoproteolysis (50% cleaved/40 min), yet this activity was lacking in each mutant protein (data not shown).
These results demonstrate that the wild type Gln 194 side chain is necessary for the general activation of RecA function and supports the suggestion by Pugh and Cox (24) that salt activation of RecA is "functionally mimicking the ionic interaction of the protein with DNA." In the presence of ATP and elevated salt concentrations, if 3 to 4 anions bind to the same sites within RecA as do phosphate groups on the DNA backbone (24,31), our data show that Gln 194 mediates the ATPinduced occupancy of these sites giving rise to an activated RecA-ATP-DNA (or RecA-ATP-salt) complex. In future studies it would be interesting to determine the ion occupancy of the Gln 194 mutant proteins. Our data identify Gln 194 as a NTP-binding site "␥-phosphate sensor" in that mutations at this residue disrupt only the ATP-induced increase in RecA activities and not the basal level functions or properties of the protein. For example, the low level DNA-independent ATP hydrolysis seen with wild type RecA is maintained for each of the Gln 194 mutant proteins indicating that Gln 194 is not an important component of the intrinsic RecA ATPase catalytic mechanism. In addition, Gln 194 appears not to be an important determinant of ATP binding because Asn, Glu, and Ala mutations show binding profiles similar to wild type RecA. We also show that each of the 3 mutant proteins maintains a low affinity binding to ssDNA in the absence of ATP similar to wild type RecA, and therefore, Gln 194 is not a determinant of this DNA binding property. We cannot necessarily exclude the possibility that Gln 194 interacts with DNA in the activated RecA-ATP-DNA complex. Using synthetic peptides corresponding to residues 193-212, which include the entire L2 region, Voloshin et al. (14) identified position 203 as an important determinant of DNA binding. Studies using peptides with substitutions at Gln 194 could assist in defining a potential role for this residue in DNA binding.
Although our data demonstrate the importance of Gln 194 in transmitting allosteric information within the RecA protein structure it certainly does not exclude other residues, perhaps within the L2 DNA binding region, as also participating in ATP-induced allosteric effects. We note that specific ATP-induced changes in the oligomeric structure of RecA are not blocked by mutations at Gln 194 (Fig. 4). Therefore, other residues must play important roles in mediating nucleotide-induced changes in RecA structure and/or function as the protein progresses through the catalytic cycle.