Genetics, Vol. 148, 989-1005, March 1998, Copyright © 1998

Genetic Analysis of Yeast RPA1 Reveals Its Multiple Functions in DNA Metabolism

Keiko Umezu1,a,b, Neal Sugawarac, Clark Chen2,a,b, James E. Haberc, and Richard D. Kolodnera,b
a Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, 02115,
b Charles A. Dana Division of Human Cancer Genetics, Dana-Farber Cancer Institute, Boston, Massachusetts 02115,
c Rosenstiel Center and Department of Biology, Brandeis University, Waltham, Massachusetts 02254-9110

Corresponding author: Richard D. Kolodner, Ludwig Institute for Cancer Research, UC San Diego School of Medicine, CMME-3080, 9500 Gilman Drive, La Jolla, CA 92093-0660, rkolodner{at}ucsd.edu (E-mail).

Communicating editor: L. S. SYMINGTON


*  ABSTRACT
*TOP
 *ABSTRACT
*MATERIALS AND METHODS
 *RESULTS
 *DISCUSSION
 *LITERATURE CITED

Replication protein A (RPA) is a single-stranded DNA-binding protein identified as an essential factor for SV40 DNA replication in vitro. To understand the in vivo functions of RPA, we mutagenized the Saccharomyces cerevisiae RFA1 gene and identified 19 ultraviolet light (UV) irradiation- and methyl methane sulfonate (MMS)-sensitive mutants and 5 temperature-sensitive mutants. The UV- and MMS-sensitive mutants showed up to 104 to 105 times increased sensitivity to these agents. Some of the UV- and MMS-sensitive mutants were killed by an HO-induced double-strand break at MAT. Physical analysis of recombination in one UV- and MMS-sensitive rfa1 mutant demonstrated that it was defective for mating type switching and single-strand annealing recombination. Two temperature-sensitive mutants were characterized in detail, and at the restrictive temperature were found to have an arrest phenotype and DNA content indicative of incomplete DNA replication. DNA sequence analysis indicated that most of the mutations altered amino acids that were conserved between yeast, human, and Xenopus RPA1. Taken together, we conclude that RPA1 has multiple roles in vivo and functions in DNA replication, repair, and recombination, like the single-stranded DNA-binding proteins of bacteria and phages.

REPLICATION Protein A [RPA, also known as Replication Factor A (RFA) or human SSB],3 is a single-stranded DNA- (ssDNA-) binding protein that was initially identified as an essential factor for replication of SV40-ori containing DNA in vitro and is likely to be the eukaryotic equivalent of the Escherichia coli SSB protein required for most aspects of bacterial DNA metabolism (STILLMAN 1989 Down; TSURIMOTO et al. 1989; KENNY et al. 1990 Down; COLLINS and KELLY 1991 Down; LEE et al. 1991 Down; DORNREITER et al. 1992 Down; BOCHKAREV et al. 1997 Down). Numerous biochemical studies have supported this point of view. Analysis of RPA in the SV40 replication system revealed that it is involved in both the initiation and elongation phases of DNA replication (STILLMAN 1989 Down; TSURIMOTO and STILLMAN 1989 Down; KENNY et al. 1990 Down; COLLINS and KELLY 1991 Down; LEE et al. 1991 Down; DORNREITER et al. 1992 Down). RPA is also involved in other aspects of DNA metabolism. RPA is required for in vitro reconstitution of nucleotide excision repair (COVERLEY et al. 1991 Down; ABOUSSEKHRA et al. 1995 Down; KAZANTSEV et al. 1996 Down) and its physical association with and functional stimulation of XPA, XPG, and XPF, along with its ability to bind to UV-damaged DNA, further confirm the importance of RPA in nucleotide excision repair (HE et al. 1995 Down; LEE et al. 1995 Down; LI et al. 1995 Down; MATSUDA et al. 1995 Down; BURNS et al. 1996 Down; MATSUNAGA et al. 1996 Down). Antibody depletion and in vitro reconstitution studies have demonstrated that RPA is required for mismatch repair (LIN et al. 1997). Finally, RPA stimulates the in vitro strand exchange activities of Saccharomyces cerevisiae SEP1 (Strand Exchange Protein), human HPP1 (Homologous Pairing Protein), and RAD51 (a eukaryotic homologue of the E. coli RecA) (HEYER et al. 1990 Down; MOORE et al. 1991 Down; ALANI et al. 1992 Down; SHINOHARA et al. 1992 Down; OGAWA et al. 1993 Down; SUNG 1994 Down, SUNG 1997 Down; SUGIYAMA et al. 1997 Down), and human RPA physically interacts with the human RAD52 gene product via RPA2 (PARK et al. 1996 Down) consistent with a role in genetic recombination. In sum, good biochemical evidence exists that RPA plays a crucial role in DNA replication, repair, and recombination.

RPA is an evolutionarily conserved protein consisting of three subunits: RPA1 (Mr 70,000), RPA2 (Mr 36,000), and RPA3 (Mr 14,000). Of these three subunits, RPA1 is the best characterized biochemically. While most biochemical characterization of RPA1 has been done using human RPA1, extrapolation of these results to other eukaryotic RPAs seems justified given the high degree of conservation among the various RPA homologues (HEYER et al. 1990 Down; BRILL and STILLMAN 1991 Down; ERDILE et al. 1991 Down; ADACHI and LAEMMLI 1992 Down; BROWN et al. 1994 Down; ISHIAI et al. 1996 Down). Functional dissection of human RPA1 has identified three distinct domains. The N-terminal 100 amino acids of human RPA1 are required for stimulation of DNA polymerase alpha (KIM et al. 1996 Down). The ssDNA-binding activity of human RPA1 resides between amino acid residues 175–420 (GOMES and WOLD 1995 Down; KIM et al. 1996 Down; LIN et al. 1996 Down). X-ray crystallographic analysis of this region suggested that it is comprised of two structurally homologous subdomains oriented in tandem (BOCHKAREV et al. 1997 Down). The overall structure of the RPA1 subdomains appears similar to those of previously solved ssDNA-binding proteins, T4 gp32 and f1 gene V protein (BOCHKAREV et al. 1997 Down). The C-terminal 100 amino acids of human RPA1 are required for binding RPA2 and RPA3 to form the RPA heterotrimer (GOMES and WOLD 1996 Down; LIN et al. 1996 Down). Located within the C-terminal third of RPA1 is a putative C4 zinc finger motif conserved among all eukaryotic homologues (ERDILE et al. 1991 Down; ADACHI and LAEMMLI 1992 Down; BROWN et al. 1994 Down; ISHIAI et al. 1996 Down), and this region of RPA1 appears to be required for DNA replication and mismatch repair but not nucleotide excision repair (LIN et al. 1996 Down; LIN et al. 1997).

While RPA1 alone binds to ssDNA with high affinity (BRILL and STILLMAN 1989 Down; WOLD et al. 1989 Down; KENNY et al. 1990 Down), the complete holocomplex is required for DNA replication in vitro (ERDILE et al. 1991 Down). Consistent with this observation, all three subunits of RPA are required for viability in S. cerevisiae (HEYER et al. 1990 Down; BRILL and STILLMAN 1991 Down). Although the functions of RPA2 and RPA3 remain to be elucidated, recently it was shown that the minimal regions of RPA2 and RPA3 necessary to maintain viability in S. cerevisiae bear weak sequence similarity to the E. coli SSB and to the two ssDNA-binding subdomains of RPA1. Based on this observation, it was proposed that the four ssDNA-binding subdomains of RPA function like the homotetrameric E. coli SSB (MANIAR et al. 1997 Down). Consistent with this hypothesis is the observation that S. cerevisiae RPA2 has contacts with ssDNA (PHILIPOVA et al. 1996 Down). Also consistent is the isolation of S. cerevisiae RFA2 and RFA3 alleles that are defective in DNA replication (SANTOCANALE et al. 1995 Down; MANIAR et al. 1997 Down). In addition to a role in ssDNA binding, RPA2 has been implicated as the regulatory subunit of the RPA heterotrimer (DIN et al. 1990 Down; DUTTA and STILLMAN 1992 Down; FOTEDAR and ROBERTS 1992 Down; CARDOSO et al. 1993 Down; LIU and WEAVER 1993 Down; CARTY et al. 1994 Down).

While there is considerable biochemical data suggesting that RPA functions in a variety of DNA metabolic processes, only limited genetic analysis has been performed to elucidate its roles in vivo. Initial studies using rfa1 null alleles reported that RFA1 is an essential gene and demonstrated that rfa1 mutations caused a terminal arrest phenotype consistent with a defect in DNA replication (HEYER et al. 1990 Down; BRILL and STILLMAN 1991 Down). One subsequent study reported the construction of two-amino-acid insertion mutations in RFA1, some of which caused weak temperature sensitivity, weak UV-sensitivity, and a modest defect in mitotic intrachromosomal recombination (LONGHESE et al. 1994 Down). Another study reported an rfa1 mutation (rfa1-44) that caused a defect in HO-endonuclease-induced plasmid-to-chromosome gene conversion and sensitivity to UV and X rays (FIRMENICH et al. 1995 Down). Overexpression of RAD52 suppressed the rfa1-44 mutation, providing evidence that RPA and RAD52 interact (FIRMENICH et al. 1995 Down). Subsequent studies of mating type switching using physical assays of recombination detected only an apparant 50% failure in recombination caused by the rfa1-44 allele (N. SUGAWARA and J. E. HABER, unpublished data). A third rfa1 mutation was isolated in a screen for suppressors of the defect in direct repeat recombination in rad1 rad52 double mutants, and this rfa1 allele by itself was found to cause some defects in genetic recombination (SMITH and ROTHSTEIN 1995 Down). Other studies of RFA1 have suggested a role for RPA in checkpoint control (BRUSH et al. 1996 Down; LONGHESE et al. 1996 Down; PARKER et al. 1997 Down) and have suggested that RPA could serve as a DNA damage sensor (BRUSH et al. 1996 Down).

While previous studies described the first rfa1 mutants, they either screened for a single phenotype and identified a single mutant (FIRMENICH et al. 1995 Down; SMITH and ROTHSTEIN 1995 Down), or they isolated a small number of mutants and then tested them for significant phenotypes (HEYER et al. 1990 Down; BRILL and STILLMAN 1991 Down; LONGHESE et al. 1994 Down). Consequently, they may not have been capable of revealing the wide variety of metabolic defects that could be caused by mutations in the RFA1 gene. In the present study, we focused on randomly mutagenizing the RFA1 gene and systematically screened for mutations that confer a temperature-sensitive (ts) and UV- or MMS-sensitive phenotype in order to isolate a variety of alleles. We isolated 24 rfa1 alleles and characterized 19 DNA-damage-sensitive mutants and two ts mutants in greater detail.


*  MATERIALS AND METHODS
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
*RESULTS
 *DISCUSSION
 *LITERATURE CITED

Media:
E. coli strains were grown in luria bertani (LB) medium (MILLER 1972 Down), which was supplemented with 100 µg/ml ampicillin when required. SOC medium (SAMBROOK et al. 1989 Down) was used for incubation of the cells after electro/transformation. Complex glucose [yeast extract reptone adenine dextrose (YPD)], sporulation, synthetic complete (SC), and various drop-out media for growth of yeast strains were made as previously described (SHERMAN et al. 1983 Down). Adenine sulfate was added to YPD at 0.004% (YPAD) when indicated. 5-Fluoro-orotic acid (5-FOA) was used as described (ROSE et al. 1990 Down). Plates containing Methyl Methane Sulfonate (MMS, Kodak, Rochester, NY) were used within two days after preparing them. For HO-induction experiments designed to test for sensitivity to HO expression, cells were grown in SC medium lacking glucose, uracil, and leucine and containing 3% glycerol and 2% sodium lactate (pH 5.5). To induce the GAL-HO gene, galactose (2%) was added to liquid cell cultures grown in this medium, or the culture was spotted onto solid medium lacking glucose, uracil, and leucine and containing 2% galactose. For analysis of HO-induced mating type switching, cells were propagated in yeast extract peptone-lactate medium and induced with galactose as previously described (CONNOLLY et al. 1988 Down).

S. cerevisiae and E. coli strains:
Strain YSB89 (MATa/MAT{alpha} ade2-1/ade2-1 ura3-1/ura3-1 his3-11,15/his3-11,15 trp1-1/trp1-1 leu2-3,112/leu2-3,112 can1-100/can1-100 rfa1::TRP1/RFA1) derived from the parental strain W303 was kindly provided by Dr. B. STILLMAN (Cold Spring Harbor Laboratory) (BRILL and STILLMAN 1991 Down). Strains RKY 2102 [MATa ade2-1 ura3-1 his3-11 trp1-1 leu2-3,112 can1-100 rfa1::TRP1 (pRPA1 URA3 RFA1)] and RKY 1900 [MATa ade2-1 ura3-1 his3-11 trp1-1 leu2-3,112 can1-100 rfa1::TRP1 (pKU1 LEU2 RFA1)] were constructed by transforming YSB89 with plasmids pRPA1 and pKU1 (see below), respectively, sporulating the strain, and then identifying an appropriate spore clone after tetrad dissection. The isogenic wild-type strain W303-1A was obtained as a spore clone from YSB89. To obtain the strain used for analysis of single-strand annealing, the W303 strain containing the rfa1-t11 allele was crossed to tNS1373M [ho HML{alpha} leu2 MAT{alpha}-inc hmr-3{Delta} mal2 trp1 thr4 GAL+ ura3-Nco-pUC-HOcs-{lambda}H3(2.3 kb)-URA3 GAL::HO at THR4] (SUGAWARA and HABER 1992 Down) to obtain rfa1-t11 segregants that were ura3 leu2 mat {alpha}-inc and possessed GAL-HO integrated at the THR4 locus. The resulting strain was then transformed with pNSU208 (see below). The E. coli strains DH5 and DH5{alpha} were used to amplify and manipulate all plasmids described in this study.

Plasmids:
The plasmid pRPA1 (HEYER et al. 1990 Down) contains a 3.4-kb Sau3A insert carrying the RFA1 gene in the BamHI site of the ARS1 CEN4 URA3 vector YCp50. Plasmid pKU1 was constructed by subcloning the 3.1-kb Sal I-HindIII fragment containing the RFA1 gene from the plasmid pRPA1 into the corresponding sites of the ARSH4 CEN6 LEU2 vector pRS415. A series of plasmids for rfa1 allele replacement pKU2 was constructed by inserting the 3.1-kb Sal I-BamHI fragment of pKU1 derivatives carrying the rfa1 mutant alleles into the corresponding sites of the URA3 integration plasmid pRS406. Plasmids pKU1 and pKU2 carrying the rfa1 mutants are indicated by hyphenating allele names. The GAL-HO plasmids pJH132 (GAL-HO URA3 ARS1 CEN4) and pJH727 (GAL1-HO LEU2 ARS1 CEN4) have been described ( JENSEN and HERSKOWITZ 1984 Down; WHITE and HABER 1990 Down). The plasmid pNSU208 was constructed by inserting the EcoRV-BssHII fragment from the lacZ sequence of pJF3 into the HindIII site of pNR16 (FISHMAN-LOBELL et al. 1992 Down).

Genetic techniques:
E. coli strains were transformed with DNA by electroporation using Gene Pulser (Bio-Rad, Richmond, CA). Standard methods for yeast genetics were essentially according to ROSE et al. 1990 Down. Transformation of yeast cells was performed using the lithium acetate procedure as described (GIETZ et al. 1992 Down). Plasmid DNA was isolated as described (ROSE et al. 1990 Down). Allele replacement was performed by standard methods involving transformation with pKU2 series plasmids that had been linearized at a site in the RFA1 gene by digestion with NheI.

RFA1 mutagenesis:
Mutagenesis of the RFA1 gene was performed by PCR based on the technique of MUHLRAD et al. 1992 Down, except that standard PCR conditions were used. The error rate of Taq polymerase is in the range of 0.1 x 10-4 to 2 x 10-4 per nucleotide under standard PCR conditions (CADWELL and JOYCE 1994 Down), and at this error rate we estimated that 12 cycles of standard PCR over a region of about 2 kb of the RFA1 open reading frame (ORF) was sufficient to generate a diverse mutation library. PCR primers were designed to amplify the 2.8-kb region of pKU1 containing the RFA1 open reading frame and the flanking sequences on both sides of the open reading frame. The primers 5'-ATGGTGCATGCAAGGAGATGGC and 5'-CACTTTATGCTTCCGGCTCCTA, which were synthesized by Molecular Biology Core Facility, Dana-Farber Cancer Institute, Boston, MA, were complementary to vector sequences located 180 nucleotides upstream and downstream from the unique MluI and HindIII sites in the vector, respectively. The MluI and HindIII sites are located 165 bp upstream from the initiation codon and 252 bp downstream from the termination codon of the RFA1 open reading frame, respectively. PCR mixtures (25 µl) contained 1.25 units of Taq DNA polymerase (Perkin Elmer, Norwalk, CT), 10 ng of template DNA (pKU1 linearized at the unique ClaI site in the LEU2 gene), 1 µM of each primer, 200 µM each dNTP, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, and 0.001% (w/v) gelatin. PCR was performed in a Perkin Elmer 9600 utilizing an initial incubation at 95° for 5 min, followed by 12 cycles of 30 sec at 95°, 30 sec at 45°, and 90 sec at 72°. Twenty independent reaction mixtures were combined (total 500 µl) and purified using a QIAquick Spin Column (QIAGEN, Santa Clarita, CA). The PCR product and the 6.8-kb MluI-HindIII fragment of pKU1 (the vector fragment of pKU1 obtained by digestion with MluI-HindIII, followed by purification by agarose gel electrophoresis to remove the 2.4-kb RFA1-containing fragment) were mixed in a 1:1 molar ratio and cotransformed into RKY 2102. Transformants were selected on SC plates lacking Leu and containing Ura and then replica-plated onto 5-FOA plates to eliminate pRPA1 by plasmid shuffling (BOEKE et al. 1987 Down). These 5-FOA plates were used as master plates for screening of rfa1 mutants. Selection and growth of transformants during this mutagenesis procedure were at 26°.

Screening of rfa1 mutants:
To screen candidate rfa1 mutants for those having temperature-sensitive (ts) growth, UV-sensitive (UVS), and MMS-sensitive (MMSS) phenotypes, each 5-FOA master plate was replica-plated onto a set of four YPD plates; 7.5 µM of erythrosine B (Sigma, St. Louis, MO) was included in the plates to stain nonviable cells (BONNEU et al. 1991 Down). In addition, MMS (0.01%) was included in one of these four plates. In some cases, adenine sulfate (YPAD) was added to suppress coloring due to the ade2 mutation and to make the red staining by erythrosine B more visible (BONNEU et al. 1991 Down). One of the YPD (or YPAD) plates was irradiated with 50 J/m2 of UV using a Stratalinker 1800 (Stratagene, La Jolla, CA) and incubated at 26° along with the YPD-MMS plate and an untreated YPD plate that served as a control. In addition, one YPD plate was incubated at 37°. After incubation for 3 days, the growth and color of the colonies on the 37°, UV, and MMS plates were compared to the control plates, and the colonies showing a growth defect or reddish color were recovered as candidate mutants and streaked for single colonies on YPD or YPAD plates containing erythrosine B. A colony from each candidate mutant was retested using the semiquantitative assays described below.

Semiquantitative assays:
Cells from a single colony were streaked on three YPD plates. One plate was incubated at 26° and served as a control. One plate was irradiated with 50 J/m2 of UV and incubated at 26°. And the third plate was incubated at 37°. Cells from the same colony were also picked into sterile water and fourfold serial dilutions were prepared. Five-microliter aliquots of each dilution were spotted onto three YPD plates, one of which contained 0.01% MMS and another of which was irradiated with 50 J/m2 of UV. These three plates were incubated at 26°. After incubation for 2 days, the plating efficiency and extent of the growth on each plate compared to the control plates were classified as follows: for UV and MMS sensitivity: s, 10- to 102-fold decrease in plating efficiency; ±, decreased <10-fold. For temperature-sensitive growth: ss, no or little growth; s, very small colonies (<10% the size of the colonies on the control plates); ±, smaller colonies (10 to 50% the size of the colonies on the control plates).

Quantitative UV survival tests:
Overnight cultures were inoculated into liquid YPAD and grown to early logarithmic phase (2–5 x 106 cells/ml). The cells were harvested by centrifugation, suspended in sterile water, recentrifuged, and resuspended in sterile water at 1 x 106 cells/ml, followed by brief sonication to disrupt aggregates. Serial dilutions were prepared in sterile water, aliquots were spread on YPD plates, and the plates were UV-irradiated as indicated. The plates were then incubated at 26° in the dark for 4 days before counting colonies.

Quantitative MMS survival tests:
Cells were prepared as described under quantitative UV survival tests to obtain a cell suspension containing 1 x 106 cells/ml. MMS was added to 0.5 ml of cell suspension at the indicated concentrations followed by incubation for 40 min at room temperature. An equal volume of freshly prepared 10% (wt/vol) NaS2O3 was added, and serial dilutions of the cells were prepared in sterile water and spread on YPD plates. Colonies were counted after incubation at 26° for 4 days.

Flow cytometry:
Cells were grown in liquid YPAD to early logarithmic phase (2–5 x 106 cells/ml) at 26°, and then the culture was divided into two portions: one portion was incubated at 26° and the other portion was incubated at 37°. Cells were withdrawn at each indicated time point and prepared for fluorescence-activated cell sorting (FACS) analysis as previously described ( JOHNSON and KOLODNER 1995 Down). The resulting samples were analyzed using a FACScan flow cytometer with the CellFIT cell cycle analysis program (version 2.01.2, Becton Dickinson, Franklin Lakes, NJ) by the Core Flow Cytometry Facility, Dana-Farber Cancer Institute. A portion of each sample also was examined by light microscopy.

DNA sequencing:
The entire region corresponding to the RFA1 open reading frame in mutant pKU1 plasmids was sequenced with Taq DNA polymerase and dye terminators using an ABI 373 DNA sequencer (Applied Biosystems, Foster City, CA) by Molecular Biology Core Facility, Dana-Farber Cancer Institute. Six sequencing primers were used and were synthesized by the Molecular Biology Core Facility: 5'-GGCGAAACCAGCAAGAAGAC, 5'-CTCAGAGCATCCAAATGAAACC, 5'-GAAGCCAAAGTATACTATGTATC, 5'-TAAAGGTGTTCGTGTGACGGA, and 5'-ATTTTGCATATCCTGCCTGTTC were complementary to the transcribed strand of RFA1 at nucleotides -39 to -20, 372 to -393, 751 to -773, 1131 to -1151 and 1701 to -1722, respectively, and 5'-CTCATATGTTACATAGATTAAATAG was complementary to the nontranscribed strand of RFA1 at nucleotides 1886 to 1911 (numbering is from the first base of the initiation codon, which is 1) (HEYER et al. 1990 Down).

Physical analysis of mating type switching and single-strand annealing:
Strains W303-1A and an isogenic derivative containing the UV S, MMSS rfa1-t11 allele were transformed with pJH727 containing GAL1::HO CEN4 LEU2. Galactose inductions were carried out as described previously (CONNOLLY et al. 1988 Down). Briefly, the strains were grown to 107 cells/ml in yeast extract peptone-lactate medium. At time zero and subsequent time points, DNA was extracted from aliquots using a glass bead protocol (CONNOLLY et al. 1988 Down). The strains were induced by the addition of galactose (2%, w/v) for 30 min, followed by the addition of glucose (2% w/v) to down-regulate the expression of GAL::HO. At 0 hr and 5 hr aliquots of cells were diluted in sterile H2O and plated on YPD. Colonies were later scored for their phenotypes. DNA samples were digested with StyI, electrophoresed through agarose gels, and analyzed by Southern blotting using a MAT-distal probe (pJH364) (WHITE and HABER 1990 Down). A Molecular Dynamics (Sunnyvale, CA) PhosphorImager was utilized to measure band intensities. Single-strand annealing was assayed using the rfa1-t11 derivative of tNS1373M essentially as described above for the physical analysis of mating type switching except for the following procedural modifications. Glucose was not added to down-regulate the expression of GAL::HO. The DNA was digested with EcoRI, blotted, and probed with the Bsu36-SacI fragment from lacZ to detect the plasmid products of HO cleavage and single-strand annealing. Colonies were grown on YPD and replica-plated onto media lacking leucine to assay for plasmid loss after induction of HO endonuclease.


*  RESULTS
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
 *RESULTS
*DISCUSSION
 *LITERATURE CITED

Isolation of rfa1 mutants:
We have attempted to systematically analyze the in vivo functions of RPA in DNA metabolism by screening for rfa1 mutants that confer ts, UV S, or MMSS phenotypes. Ts mutants were selected to identify mutations causing defects in DNA replication. UV S mutants were selected to identify mutations causing defects in nucleotide excision repair and/or recombinational repair. And MMSS mutants were selected to identify mutations that cause defects in recombination as well as possibly defects in other types of DNA repair (ESPOSITO and WAGSTAFF 1981 Down; PETES et al. 1991 Down). The mutants isolated in these screens have subsequently been tested for mutator phenotypes, and the results of these studies will be described elsewhere.

The yeast strain RKY2102 was cotransformed with RFA1 fragments that had been amplified by PCR under mutagenic conditions and pRS415 derivative vector fragments, resulting in reconstruction of an RPA1 plasmid by in vivo homologous recombination. We then screened 15,000 Leu+ 5-FOA-resistant transformants for ts, UVS, and MMSS phenotypes, first using erythrosine B staining as a growth defect indicator (the first screening) and second using semiquantitative measurements of the expected phenotypes. Fifty-four candidate mutants were obtained for further study, some of which had more than one phenotype. No mutants were obtained in control experiments in which RFA1 was not amplified by PCR prior to the transformation step.

The resulting candidate mutant plasmids were retested by isolating plasmid DNA from each mutant and transforming it back into RKY2102 followed by plasmid shuffling. Nineteen out of 22 of the UV S and/or MMSS mutant plasmids conferred a similar degree of sensitivity to that of the original mutants from which the plasmid had been isolated. The sensitivity of all 19 mutants was suppressed in the presence of pRPA1 carrying the wild-type RFA1 gene, indicating that the mutants were all recessive. After plasmid rescue, only 5 out of 10 ts mutant plasmids tested conferred a ts phenotype. All 5 of these ts mutants were recessive to wild type. In total, 24 mutants were obtained and 21 were studied further; specific examples of the analysis of these mutants are presented below, and a summary of the properties of all of the mutants, including quantitative data, are presented in Figure 8 at the end of this article.



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  		Figure 1. —Survival of representative mutants treated with either UV-irradiation or MMS. Quantitative UV and MMS survival tests were performed as described in materials and methods. The number shown next to each symbol indicates the mutant allele tested. The wild-type RFA1 control strain, RKY1900, carries the RFA1 gene on pKU1 and has the same rfa1 deletion mutation on the chromosome as the rfa1 mutant strains tested; all of the rfa1 alleles examined were present on a plasmid in this experiment.



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  		Figure 2. —Sensitivity of UVS and MMSS mutants to HO endonuclease. The indicated mutant strains transformed with the GAL-HO plasmid pJH132 were grown in SC medium lacking glucose, uracil, and leucine and containing 3% glycerol and 2% sodium lactate (pH 5.5) to early logarithmic phase. Approximately 7 x 104 cells from each culture were spotted in duplicate onto an SC plate lacking glucose, uracil, and leucine and containing 2% galactose to induce the GAL-HO gene (left panel) or were spotted onto an SC plate lacking uracil and leucine as control (right panel). The plates were then incubated at 26° for 4 days. As in Figure 1, RKY1900 was used as a wild-type control strain.



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  		Figure 3. —RFA1 is required for mating type switching after induction of HO endonuclease. An rfa1-t11 mutant containing the rfa1 allele at the chromosomal locus and a wild-type strain were induced to switch mating type by expression of a GAL::HO fusion. DNA was extracted at the time points shown, digested with StyI, electrophoresed under neutral conditions, blotted and hybridized with a MAT-specific probe (hatched box). At 0 hr the probe hybridizes to the MATa fragment (0.93 kb) and the MATdistal fragment (4.3 kb). After GAL::HO expression a smaller HO-cleaved fragment (0.72 kb) appears, followed at later time points by the appearance of a MATa product band (1.88 kb). This band is larger because it lacks a StyI site in the Ya sequence that is present in Ya. In the rfa1 mutant the product band is faint but detectable.



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  		Figure 4. —RFA1 is required for efficient single-strand annealing. (A) The plasmid pNSU208 (15 kb) contains two 0.24-kb direct repeats composed of lacZ sequences with an HO cut site in between. Cleavage by HO endonuclease is followed by 5' to 3' degradation of DNA leaving a 3' single-stranded tail. The complementary lacZ sequences can anneal and be processed to yield a deletion product. (B) Single-strand annealing was initiated in vivo when a double-strand break was created by induction of the GAL::HO endonuclease gene in an rfa1-t11 mutant containing the rfa1 allele at the chromosomal locus and a wild-type strain. Cleavage of the 9.4-kb pNSU208 EcoRI fragment by HO endonuclease resulted in the production of 6.2- and 3.2-kb fragments by 0.5 hr and yielded a 7.8-kb product fragment in the wild-type strain. The amount of product formed was greatly reduced in the rfa1-t11 mutant.



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  		Figure 5. —Some rfa1 mutants display a strong temperature-sensitive phenotype. The indicated mutant and wild-type strains were streaked on two YPD plates that were incubated at 26° (left panel) and at 37° (right panel) for 3 days, respectively. The allele number is indicated next to each streak. When the allele designation is followed by -c, it indicates the allele at the chromosomal locus, whereas -p indicates the allele carried on an ARS CEN plasmid. For wild-type control strains, RKY 1900 was used as wt-p and W303-1A was used as wt-c.



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  		Figure 6. —FACS analysis of DNA content. Cells were grown in liquid YPAD to early logarithmic phase at 26°, and then the culture was divided into two portions: one portion was incubated at 26° and the other portion was incubated at 37°. Cells were withdrawn after 4 hr, stained with propidium iodide, and analyzed using a FACScan flow cytometer. The results obtained with the rfa1-t33 allele integrated at the chromosomal locus and the wild-type strain W303-1A are shown here. Identical results were obtained when similar analysis was performed with the rfa1-t6 allele at the chromosomal location.



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  		Figure 7. —Morphological analysis of wild-type and rfa1-t33 cells at the restrictive temperature. A portion of the culture used in the experiments shown here was withdrawn at each indicated time point and examined by light microscopy. Cells in which the bud size was less than one-third the size of the mother cell were classified as "small buds," while the cells harboring larger buds were classified as "large buds." Cells having multiple buds or protruded buds were classified as "other." The results obtained with the rfa1-t33 allele integrated at the chromosomal locus and the wild-type strain W303-1A are shown here. Identical results were obtained when similar analysis was performed with the rfa1-t6 allele at the chromosomal location.



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  		Figure 8. —Summary of the phenotypes caused by rfa1 mutations and illustration of the positions of the mutations relative to the positions of known RFA1 structural features. The coordinates of the different functional regions are taken from the papers discussed in the introduction and discussion (ERDILE et al. 1991 Down; ADACHI and LAEMMLI 1992 Down; BROWN et al. 1994 Down; GOMES and WOLD 1995 Down, GOMES and WOLD 1996 Down; ISHIAI et al. 1996 Down; KIM et al. 1996 Down; LIN et al. 1996 Down; PARK et al. 1996 Down; PHILIPOVA et al. 1996 Down; BOCHKAREV et al. 1997 Down). The positions of the indicated amino acid changes are given relative to the structural features of the protein and are based on the following mutational data: Ile14Ser, T41G; Phe15Leu, T43C; Met49Thr, T146C; Lys45Glu, A133G; Asp121Gly, A362G; Arg109Cys, C325T; Phe124Ser, T371C; Glu562Val, A1685T; Ser373Pro, T1117C; Gly207Arg, G619A; Tyr255Cys, A764G; Leu221Pro, T662C; Phe222Ser, T665C; His274Arg, A821G; Ser230Pro, T688C; Ser351Pro, T1051C; Leu602Ser, T1805C; Ser391Pro, T1171C; Leu410Ser, T1229C; Ser569Cys, A1705T; Lys493Glu, A1477G; Lys494Glu, A1480G; Phe537Ile, T1609A. The mutational changes are annotated in the following manner: open triangle, residue mutated is not conserved among S. cerevisiae, human, Drosophila, and Xenopus RPA1; closed triangle, residue mutated is conserved among S. cerevisiae, human, Drosophila, and Xenopus RPA1. In addition, the following silent nucleotide changes were found: rfa1-t22, A255T; rfa1-t69, T195C; rfa1-t23, A630G; rfa1-t63, C1074G; rfa1-t155, T1533C. UV- and MMS-sensitivity were determined at a fixed dose of either 75 J/m2 of UV-irradiation or 40 min of incubation in 0.5% MMS as described in materials and methods and in Figure 1: the values given are the fold reduction in survival relative to the wild-type strain. (The fraction of wild-type cells that survived UV treatment was 0.21 and the fraction of wild-type cells that survived MMS treatment was 0.34.) HO sensitivity summarizes the data presented in Figure 2: —, average of >20 colonies per patch survive upon HO induction (wild-type level); s, average of 10–20 colonies per patch survive upon HO induction; ss, average of 6–10 colonies per patch survive upon HO induction; sss, average of 0–5 colonies per patch survive upon HO induction; nt, not tested. Temperature sensitivity refers to whether a consistent temperature-sensitive phenotype like that illustrated in Figure 5 was seen as defined in materials and methods under Semiquantitative assays. The doubling times presented were determined by growing the indicated strains in liquid YPD medium at 26° and monitoring cell growth both by counting cell numbers and by plating onto YPD plates to determine the number of viable cells present. All of the data summarized in this figure were generated with strains in which the rfa1 alleles were present on a plasmid.

UV S and MMSS mutants:
Nineteen mutants showing increased sensitivity to UV-irradiation and/or MMS were identified and analyzed in greater detail using quantitative tests. Figure 1 shows the survival curves obtained after treatment of representative mutants with a range of doses of either UV-irradiation or MMS, and the survival of each of these mutants after treatment with a fixed dose of either UV-irradiation or MMS is presented in Figure 8. All experiments were performed using rfa1{Delta} mutant strains (RKY 2102 derivatives) in which the mutant rfa1 allele of interest was present in the strain on an ARS CEN plasmid. In addition, control experiments were performed using RKY 2102 and RKY 1900 derivatives carrying the wild-type RFA1 gene on an ARS CEN plasmid. Selected mutations were also transferred to the chromosomal RFA1 locus and similarly analyzed to ensure that the plasmid-born alleles and chromosomal alleles behaved similarly.

All of the mutants obtained were sensitive to both UV-irradiation and MMS, even though the initial qualitative tests suggested the existence of mutants that were sensitive to killing by only one of the two agents. The mutants showed a wide range of sensitivity to both UV-irradiation and MMS at the doses tested; survival of mutants relative to the wild-type strain ranged from 10-3 to 0.5 for UV-irradiation and from 10-4 to 0.7 for MMS (Figure 8). There was a direct correlation between the degree of sensitivity to UV-irradiation and to MMS for each mutant. An interesting feature of the data is that the weakly and moderately UV S alleles showed similar sensitivity to MMS, whereas the strongly UV S alleles showed an even greater sensitivity to MMS; this is more apparent when the data presented in Figure 8 are reranked in order of increasing sensitivity.

The experiments discussed above (Figure 1 and Figure 8) were performed with strains carrying the rfa1 allele on an ARS CEN vector. Although we obtained the same results when either YPAD or Leu drop-out media that select for the marker on the plasmid (LEU2) was used, loss of the plasmid could conceivably have affected the results. To eliminate this possibility, the rfa1-t11 allele was integrated at the chromosome RFA1 locus, and the UV-sensitivity and MMS-sensitivity of the resulting strain were determined. Survival of this strain to killing by UV-irradiation and MMS was 3.85 x 10-4 and 1.02 x 10-4, respectively, compared to the wild-type parental strain, which was similar to that obtained with rfa1-t11 allele on an ARS CEN plasmid at the same doses of UV-irradiation and MMS (Figure 8). Similar results were obtained with the rfa1-t49 allele as well as three additional alleles that will be described elsewhere.

Some of the mutant strains, especially the strongly UV S and MMSS mutants, had an increased doubling time compared to the wild-type strains tested (Figure 8). This could be due to failure to repair endogenous DNA damage in these mutants resulting in triggering of a DNA damage checkpoint. Alternately, these mutant strains might also have defects in DNA replication that cause reduced growth rates. To examine these possibilities, we analyzed early logarithmic phase cells of strains carrying either the rfa1-t11 chromosomal allele or the rfa1-t11 plasmid allele using both FACS and light microscopy. Compared to the wild-type control strain (RKY 2102), both mutant strains showed no significant increase in the proportion of cells present in a specific phase of the cell cycle such as S phase or G2 phase (data not shown). This suggests that the rfa1-t11 allele does not cause a defect in DNA replication or trigger a DNA damage checkpoint.

Sensitivity of UVS and MMSS mutants to HO endonuclease:
We anticipated that mutants that were sensitive to killing by both UV-irradiation and MMS might also have defects in genetic recombination because both UV-irradiation and MMS are known to produce damage in DNA that can be repaired by genetic recombination (PETES et al. 1991 Down). To examine whether the UV S and MMSS mutants had such defects, we induced double-strand breaks at MAT by galactose induction of HO-endonuclease and determined whether the mutants were killed by this treatment; the mutants showing very weak UV S and MMSS phenotypes were not tested. Two different types of tests were performed: one measured the plating efficiency of the mutants on plates containing galactose and the other measured the effect of transient induction of HO on survival. These tests allowed the identification of three classes of mutants.

The transformation efficiency of mutants carrying the rfa1-t21, rfa1-t155, and rfa1-t18 alleles with pJH132 (pGAL-HO) was much lower than with the other strains examined when the transformants were selected on plates containing glucose. The pJH132-transformed cells had growth defects even on glucose plates without galactose and were unable to survive on galactose plates (Figure 2). This suggests that these mutants were extremely sensitive to double-strand breaks and that either the extremely low level of HO expression in the presence of glucose is sufficient to kill some of the cells or that HO expression is increased in these rfa1 mutants.

Strains carrying the rfa1-t11, rfa1-t22, rfa1-t48, and rfa1-t69 alleles and the pGAL-HO plasmid showed clear differences in growth in the presence and the absence of galactose (Figure 2). These strains grew somewhat more slowly than a wild-type strain on glucose plates, and the survival of these mutant strains on galactose plates was much lower than the wild-type control strain. The strain carrying the rfa1-t11 allele was examined in greater detail by exposing the strain to galactose for different periods of time and then plating the cells onto glucose plates to measure survival. Ninety percent of the cells carrying the rfa1-t11 allele were killed after 4 hr of exposure to galactose compared to the wild-type control strain, confirming the extreme sensitivity of this mutant to even transient induction of double-strand DNA breaks at MAT (data not shown).

To further investigate the role of RPA, we monitored recombination induced by a double-strand break by Southern analysis. Mating type switching was induced in mutant and wild-type cultures by galactose induction of HO endonuclease from pJH727. After induction, DNA samples were extracted and analyzed by Southern blotting. Figure 3 shows efficient cutting at MATa by the HO endonuclease followed by the appearance of a product band (MAT{alpha}) in the wild-type control. The chromosomal rfa1-t11 mutant generates a comparable level of HO-cleaved DNA but much less product DNA. Over time the HO-cleaved DNA in the rfa1-t11 mutant disappeared, consistent with the cleaved DNA being degraded and the cells dying as was observed on transient exposure to galactose (discussed above). The MAT{alpha} product band produced in the rfa1-t11 mutant represents 4.4% of the total MATa and {alpha} sequences, compared with 56% for the wild-type strain. The kinetics of appearance of MAT{alpha} product in the rfa1-t11 mutant strain are indistinguishable from the wild-type strain. This defect in switching in the rfa1-t11 mutant was confirmed by examination of the colonies produced after 5 hr of HO-induction. In the rfa1-t11 mutant only 7% of the colonies maintaining the GAL::HO plasmid successfully switched from MATa to MAT{alpha} (55% switched in the wild-type strain). The rfa1-t11 allele is not a null allele and retains some types of RFA1 activity. Nevertheless, both the Southern blot analysis and the colony assays indicate that the rfa1-t11 mutant is highly defective in its ability to carry out double-strand break-induced recombination.

Induction of HO endonuclease had a less clear effect on strains carrying the rfa1-t49, rfa1-m51, rfa1-t23, and rfa1-t83 alleles (Figure 2), consistent with the observation that these alleles were among the moderate UV S and MMSS alleles identified. This suggests that these mutations only cause minor defects in homologous recombination, resulting in only moderate sensitivity to UV-irradiation, MMS, and HO endonuclease. Alternately, these mutations may not affect homologous recombination and may only cause defects in repair pathways like nucleotide excision repair or base excision repair.

Defects in single-strand annealing in the rfa1-t11 mutant:
Single-strand annealing (SSA) is a process in which single-stranded DNA regions are generated on each side of a double-strand break in the form of 3' tails (SUGAWARA and HABER 1992 Down). When complementary sequences on each side of the break become single stranded, they can anneal and subsequently be processed to result in a single copy, while deleting the intervening sequence. Given RFA1's involvement in mating type switching, a process mediated by double-strand break repair, we tested whether RFA1 has a role in SSA by utilizing an HO cut site situated between direct repeats of a 0.24-kb E. coli lacZ sequence carried on a centromeric plasmid. A Southern hybridization analysis of DNA extracted from a culture undergoing SSA revealed that the rfa1-t11 mutant was impaired in its ability to carry out SSA relative to a wild-type strain (Figure 4). Although a small amount of product could be formed, the mutant was 8.5 times less efficient than wild type in carrying out SSA based on a densitometric analysis of the blots. This defect was also manifested by the loss of HO endonuclease-cleaved plasmid substrate in the mutant strain. The number of colonies retaining the plasmid after induction compared to those before induction was 0.97 for wild type but only 0.12 for the rfa1-t11 mutant.

Analysis of ts rfa1 mutants:
We identified 5 ts mutants (of 10 mutants that were retested) with which it was possible to demonstrate that the ts phenotype was associated with an rfa1 mutation. Two of these mutant alleles (rfa1-t6 and rfa1-t33) were successfully transferred to the chromosomal RFA1 locus, whereas it was not possible to transfer the other three mutant alleles to the chromosomal RFA1 locus (Figure 5). As shown in Figure 6, strains carrying the rfa1-t33 and rfa1-t6 alleles at either the chromosomal locus or on an ARS CEN plasmid had a clear temperature-sensitive phenotype. Strains carrying the rfa1-t33 allele, both when integrated on the chromosome or on the ARS CEN plasmid, grew normally at the permissive temperature, while at the restrictive temperature they grew only at heavily streaked regions and formed no visible individual colonies. Strains carrying the chromosomal rfa1-t6 allele grew more slowly at the permissive temperature than strains carrying this allele on an ARS CEN plasmid. Strains carrying the chromosomal rfa1-t6 allele did not grow at all at the restrictive temperature, whereas strains carrying this allele on an ARS CEN plasmid grew only at heavily streaked regions and formed tiny colonies at the restrictive temperature. Thus, the ts phenotype caused by the integrated alleles (particularly in the case of the rfa1-t6 allele) was more severe than the plasmid-born alleles. This suggests that the reason we were not able to transfer three of the ts rfa1 alleles to the chromosomal locus was that they were lethal when integrated at the RFA1 chromosomal locus.

The ts mutants did not initially appear to be sensitive to killing by UV-irradiation or MMS when they were characterized using quantitative assays as plasmid-born alleles. However, when the rfa1-t33 and rfa1-t6 alleles were transferred to the chromosomal locus, the resulting strains became sensitive to UV-irradiation and MMS. When these alleles were present on an ARS CEN plasmid, they conferred only limited sensitivity to UV-irradiation and MMS: Survival of a strain carrying a plasmid-born rfa1-t33 allele was 0.143 and 0.116 after treatment with 75 J/m2 of UV-irradiation and 0.5% MMS, respectively, and survival of a strain carrying a plasmid-born rfa1-t6 allele was 8.27 x 10-2 and 8.4 x 10-2, respectively, which was similar to that observed for the wild-type control strain (Figure 8). However, when these alleles were transferred to the chromosomal RFA1 locus, they caused significantly increased sensitivity to both UV-irradiation and MMS at the standard doses tested: Survival of the rfa1-t33 strain after treatment with 75 J/m2 of UV-irradiation and 0.5% MMS was 1.01 x 10-2 and 1.83 x 10-3, respectively, and survival of the rfa1-t6 strain was 3.48 x 10-3 and 2.05 x 10-3, respectively. These results suggest that expression of RPA1 from the chromosome is somewhat lower than that on an ARS CEN plasmid and that repair and/or recombination is more sensitive to RPA1 levels than DNA replication in the case of the ts alleles rfa1-t6 and rfa1-t33, as compared to the repair-defective alleles rfa1-t11 and rfa1-t49 where no difference between plasmid and chromosomal alleles was observed (see above).

To characterize the ts phenotype conferred by the rfa1-t33 and rfa1-t6 alleles in more detail, we grew cultures of strains carrying these alleles at the permissive temperature, shifted logarithmically growing cultures to the nonpermissive temperature, and examined the resulting changes in cellular morphology and DNA content (Figure 6 and Figure 7). The experiments presented were performed with a wild-type control strain and a strain carrying the rfa1-t33 allele at the chromosomal locus; however, identical results were obtained with both the rfa1-t33 and rfa1-t6 alleles, regardless of whether they were on an ARS CEN plasmid or at the chromosomal RFA1 locus (data not shown).

Figure 6 shows FACS analysis of the DNA content of wild-type and rfa1-t33 cells (identical data were obtained with the rfa1-t6 allele but are not presented due to space considerations). After 4 hr at the restrictive temperature, the rfa1-t33 strain showed a clear difference in the distribution of cells having differing DNA contents compared to the wild-type strain. The rfa1-t33 strain showed a significant decrease in the proportion of cells having either a 1N or 2N DNA content and a significant increase of the proportion of cells having greater than 1N but less than 2N DNA content compared to the wild-type control strain. This effect was seen to a lesser extent after 2 hr of incubation at the nonpermissive temperature and did not increase with continued incubation beyond 4 hr (data not shown). These results suggest that most of the rfa1-t33 cells did not complete DNA replication and accumulated prior to M phase at the restrictive temperature. This was confirmed by analyzing the cells by light microscopy (Figure 7, discussed below); identical data were obtained with the rfa1-t6 allele but are not presented due to space considerations. After 4 hr at the restrictive temperature, 70% of the rfa1-t33 cells had arrested with a single large bud. After staining with DAPI, these large-budded cells were observed to have a single nucleus at the neck of the bud (data not shown). The large-budded cells appeared to have been derived from the small-budded cells presented in the culture at the time of temperature shift because the proportion of small-budded cells decreased by the same amount that the proportion of large-budded cells increased, whereas the proportion of nonbudded cells present in the culture did not appear to change. In addition, approximately 3% of the arrested rfa1-t33 cells had protruded buds or dumbbell-shaped buds like those observed after sporulation of rfa1 null mutants (HEYER et al. 1990 Down). Control experiments with the wild-type parental strain showed a normal distribution of cells at different stages of the cell cycle at both temperatures. From these results, we conclude that the ts rfa1 mutants rfa1-t33 and rfa1-t6 have defects in the elongation stage of DNA replication at the restrictive temperature, resulting in cell cycle arrest at a premitotic stage, or they arrest prior to M phase at a step that causes a block in DNA replication.

In the experiments presented in Figure 6 and Figure 7, we observed that prior to shifting to the restrictive temperature, 15–20% of the rfa1-t33 cells had 1N DNA content and a similar proportion of the cells did not have buds consistent with 15–20% of the cells in G1. After 2 and 4 hr at the restrictive temperature, the proportion of such rfa1-t33 cells did not change. This suggests that either these cells are dead or that they are unable to enter S phase and initiate DNA replication. Similar results were obtained with rfa1-t6 cells (data not shown).

DNA sequence analysis of rfa1 mutations:
To determine the nature of the rfa1 mutations causing the observed altered phenotypes, we sequenced the rfa1 gene of all 19 UV S and MMSS alleles and both ts alleles. Figure 8 and Figure 9 summarize the mutations identified and the predicted amino acid changes they cause. Every allele had at least one mutation in the RFA1 gene. Thirteen alleles had either a single nucleotide change or two changes of which one was a silent change, allowing clear identification of the causal mutation. Eight alleles had two potentially significant nucleotide changes. There were no identical alleles; however, rfa1-t155 could easily be related to rfa1-t21 by the subsequent accumulation of a second, silent nucleotide change.



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  		Figure 9. —Alignment of different RPA sequences and amino acid changes caused by rfa1 mutations. Alignment of the predicted RFA1 amino acid sequences for the ssDNA binding domains 1 and 2 of S. cerevisiae, human, Drosophila and Xenopus RFA1 (see PHILIPOVA et al. 1996 Down; BOCHKAREV et al. 1997 Down). Identities are indicated by boxes, and the positions of the amino acid changes from Figure 8 are indicated by circles and are labeled with the relevant allele numbers. S. cerevisiae and human are indicated by y and h, respectively. Domains 1 and 2 are indicated as RPA1A and RPA1B, respectively. Down-arrows indicate ssDNA-binding contacts in domain 1 and up-arrows indicate ssDNA-binding contacts in domain 2.

Twenty-three mutations were identified, and most of them changed amino acids that were conserved among yeast, human, Drosophila melanogaster, and Xenopus lavis (see Figure 8 for an analysis of the mutations, including a list of mutations affecting conserved amino acids). It is possible to ascribe significance to some of these mutations based on what is known about the structure of RFA1. Five mutations mapped in the region necessary for pol {alpha} stimulation by RFA (KIM et al. 1996 Down). Three mutations causing very weak phenotypes mapped in the region between the regions required for pol {alpha} stimulation and DNA binding. Ten mutations, including two causing a high degree of sensitivity to UV-irradiation and MMS (rfa1-t48 and rfa1-m51), mapped in either domain 1 or domain 2 of the conserved region of RFA1 required for ssDNA binding (HEYER et al. 1990 Down; GOMES and WOLD 1995 Down; PHILIPOVA et al. 1996 Down; BOCHKAREV et al. 1997 Down). The mutations rfa1-t21, rfa1-t155, and rfa1-t141 altered amino acids in the putative zinc finger of RFA1, which is not necessary for DNA-binding activity (GOMES and WOLD 1995 Down; LIN et al. 1996 Down) but could act to mediate protein-protein interactions (GALCHEVA-GARGOVA et al. 1996 Down; RODGERS et al. 1996 Down; SHEPARD et al. 1996 Down; SZABO et al. 1996 Down). Finally, at least one mutation mapped in the C-terminal region required for RPA holocomplex formation. Understanding the exact effects of each mutation on the function of RPA1 will require a detailed analysis of the biochemical properties of the mutant RPA1 proteins. However, these data do allow us to make some predictions about the functions of the different regions of RPA1, and these will be discussed below.

In the case of alleles having two nucleotide changes, it is not clear which nucleotide change is the causal mutation. However, some insights into the nature of these alleles can be obtained by comparing the properties of these alleles. The rfa1-t22 allele had two nucleotide changes, one of which in position 43 was in common with the rfa1-t47 allele that had only that change. Because the rfa1-t47 allele causes a lower degree of UV- and MMS-sensitivity than the rfa1-t22 allele, it seems likely that the second nucleotide change present in the rfa1-t22 allele (position 146) also contributes to the mutant phenotype caused by the rfa1-t22 allele. The rfa1-t11 and rfa1-t69 alleles cause a similar degree of UV- and MMS-sensitivity. This suggests that the common nucleotide change at position 133 might be the causal mutation, while the second nucleotide change in the rfa1-t69 allele at position 362 may not contribute to the mutant phenotype. One of the strong ts alleles, rfa1-t6, shared the same nucleotide change at position 1051 with the rfa1-t83 allele, which had an additional change at position 1805. Because the rfa1-t83 allele caused moderate UV- and MMS-sensitivity in addition to a ts phenotype, the latter change at position 1805 might be responsible for its UV- and MMS-sensitive phenotypes, while the former change at position 1805 might be the causal mutation for temperature sensitivity. It will be necessary, however, to use site-directed mutagenesis to construct appropriate single mutants to fully understand the nature of the alleles containing more than one potentially significant nucleotide change.

The rfa1-t33 and rfa1-t92 alleles had one nucleotide change in common, a T to C change at position 1117 that changed the serine to a proline. The rfa1-t33 allele caused a strong ts phenotype, whereas the rfa1-t92 allele just showed slight UV-sensitivity (Figure 8). These results suggest that the second mutation in rfa1-t92 suppressed the ts phenotype caused by the serine to proline change and caused the weak UV-sensitive phenotype, possibly in combination with the serine to proline change.


*  DISCUSSION
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
 *RESULTS
 *DISCUSSION
*LITERATURE CITED

In an effort to understand the role of RPA in DNA metabolism and to begin to identify regions of RPA required for different RPA-dependent processes, we have used a plasmid shuffle method to isolate random mutations in the RFA1 gene encoding the 70 kDa subunit of RPA. These mutants were then screened for ts, UV S, and MMSS, and 24 mutants having a variety of different phenotypes were obtained. In addition, we have retained a large number of other uncharacterized rfa1 mutants that will be useful for future studies. Previous studies described the first rfa1 mutants and some of the phenotypes caused by rfa1 mutations; however, these studies identified only a limited number of rfa1 mutants (HEYER et al. 1990 Down; BRILL and STILLMAN 1991 Down; LONGHESE et al. 1994 Down; FIRMENICH et al. 1995 Down; SMITH and ROTHSTEIN 1995 Down). The preliminary analysis of the rfa1 mutants described here has documented a variety of phenotypes caused by rfa1 mutations, including several causing much stronger replication and recombination defects than previously described. Based on the results obtained thus far, it is likely that we will be able to use the mutant collection described here in conjunction with considerable additional experimentation to define in greater detail different roles of RPA in DNA metabolism.

Biochemical studies of RPA have shown that it stimulates the homologous pairing activity of several different homologous pairing proteins, SEP1 (HEYER et al. 1990 Down; ALANI et al. 1992 Down), HPP1 (MOORE et al. 1991 Down), and RAD51 (SUNG 1994 Down, SUNG 1997 Down; SUGIYAMA et al. 1997 Down) and interacts with proteins required for excision repair (HE et al. 1995 Down; LEE et al. 1995 Down; LI et al. 1995 Down; MATSUDA et al. 1995 Down; MATSUNAGA et al. 1996 Down) and genetic recombination (FIRMENICH et al. 1995 Down; PARK et al. 1996 Down). Consistent with this, we were able to isolate a number of rfa1 alleles that conferred different degrees of sensitivity to UV-irradiation and MMS (Figure 1 and Figure 8). Interestingly, most of these alleles were not temperature-sensitive and did not appear to cause defects in DNA replication. Similarly, many of the mutations that caused replication defects had only limited effects on repair of UV- and MMS-induced damage to DNA. These results suggest that there are a number of ways in which mutations in RFA1 may alter the properties of RPA without changing its ability to function in DNA replication. This may reflect the possibility that the DNA and protein-protein interaction requirements for RPA1 may differ significantly between DNA replication and DNA recombination/repair. For example, the ability of RPA1 to interact with and stimulate the activity of XPA, XPF, XPG, and RAD52 (FIRMENICH et al. 1995 Down; HE et al. 1995 Down; LEE et al. 1995 Down; LI et al. 1995 Down; MATSUDA et al. 1995 Down; MATSUNAGA et al. 1996 Down; PARK et al. 1996 Down) likely reflects a function that is required for recombination and repair but not for replication. An interesting parallel in this regard is the E. coli SSB, where the ssb-113 mutation alters its DNA-binding properties so as to cause repair and recombination defects but not replication defects (MEYER and LAINE 1990 Down).

Previous genetic studies have provided evidence that RPA functions in genetic recombination (LONGHESE et al. 1994 Down; FIRMENICH et al. 1995 Down; SMITH and ROTHSTEIN 1995 Down) and interacts with at least one recombination protein. Two of these studies reported the isolation of rfa1 alleles that caused modest, less-than-10-fold reductions in direct repeat recombination (SMITH and ROTHSTEIN 1995 Down) and mitotic intrachromosomal recombination (LONGHESE et al. 1994 Down), respectively. One of these alleles also stimulated recombination between direct repeats in a rad1 rad52 double mutant (SMITH and ROTHSTEIN 1995 Down). A third study reported the isolation of an rfa1 allele (rfa1-44) that caused a strong defect in a double-strand break-induced plasmid by chromosome gene conversion (FIRMENICH et al. 1995 Down). Subsequent studies have shown that the rfa1-44 allele caused ~50% failure in mating type switching (J. E. HABER and N. SUGAWARA, unpublished data), a result that is not surprising because the mutant isolation procedure used involved induction of HO endonuclease in a MAT strain that should be lethal in the presence of mutations causing strong defects in double-strand break repair. We found that many of the UV S and MMSS mutants described here showed some degree of sensitivity to killing by HO endonuclease compared to the wild-type control strain (Figure 2), suggesting they might be highly defective in recombinational repair of double-strand breaks. More detailed analysis of one UV S and MMSS mutant (rfa1-t11) using genetic and biophysical assays that measure mating type switching showed that this allele caused over a 90% decrease in mating type switching in physical assays (Figure 3), consistent with a strong defect in double-strand break repair. The rfa1-t11 allele also caused ~90% decrease in SSA recombination; this is the first report of such an effect of an RFA allele. Interestingly, the rfa1-t11 allele was not the most HO endonuclease-sensitive, UV S, or MMSS allele we obtained (Figure 2 and Figure 8), suggesting that there are a number of mutants among our collection that are even more recombination defective than rfa1-t11. More detailed analysis of these mutants will be performed in the future to more completely determine their effects on different types of recombination.

Many of the rfa1 mutants we isolated confer a UV S phenotype. This is consistent with the view that RPA might play a direct role in the excision repair of UV damage. Such a role for RPA is based on the observations that human RPA is required for excision repair in vitro (COVERLEY et al. 1991 Down) and that RPA1 interacts with proteins required for excision repair (ABOUSSEKHRA et al. 1995 Down; HE et al. 1995 Down; LEE et al. 1995 Down; LI et al. 1995 Down; MATSUDA et al. 1995 Down; MATSUNAGA et al. 1996 Down). However, the UV S phenotypes we observed could be due to defects in either excision repair or recombinational repair of UV damage. Given the MMS-sensitivity and HO-endonuclease sensitivity of many of the UV S mutants we obtained, it would not be surprising if the UV S phenotype observed reflected a defect in recombinational repair rather than excision repair. Additional analysis will be required to distinguish between these possibilities.

RPA was originally identified because of its requirement for in vitro DNA replication, suggesting that it is an essential DNA replication factor (WOBBE et al. 1987 Down; WOLD et al. 1987 Down; FAIRMAN and STILLMAN 1988 Down), and several genetic studies have provided limited data supporting this view. Analysis of the terminal arrest phenotype of spores containing an rfa1 null allele showed that such spores germinate and arrest with either a large bud or multiple large buds and a single nucleus consistent with a lack of DNA replication (HEYER et al. 1990 Down; BRILL and STILLMAN 1991 Down). In addition, rfa1-M2 mutant cells were weakly temperature-sensitive and a higher proportion of cells held at the restrictive temperature had an S phase DNA content than wild-type cells suggestive of some defect in DNA replication (LONGHESE et al. 1994 Down). In the studies presented here we identified two rfa1 alleles (rfa1-t6 and rfa1-t33) that caused a strong temperature-sensitive phenotype (Figure 5). In both cases, 70% of the mutant cells held at the restrictive temperature arrested with a large bud having a single nucleus at the neck of the bud (data not shown), and consistent with this, 80 to 85% of the cells held at the restrictive temperature had a sub-2N DNA content. [The data for the rfa1-t33 allele are presented in Figure 6 and Figure 7; however, identical results were obtained with the rfa1-t6 allele (data not shown).] The large-budded cells appeared to have accumulated from small-budded cells (S phase cells) that progressed through the cell cycle and arrested prior to M phase because the proportion of small-budded cells decreased by the same amount as the proportion of large-budded cells increased during incubation at the restrictive temperature. In contrast, the proportion of nonbudded cells present in the culture did not appear to change during the time course of the experiment. These data are consistent with the idea that the rfa1-t6 and rfa1-t33 alleles cause a defect in the elongation stage of DNA replication resulting in the accumulation of cells prior to M phase. Alternately, these alleles could cause some other type of S-phase/G2-phase defect, in turn causing some type of inappropriate progression through the cell cycle resulting in a failure to complete DNA replication and arrest prior to M phase. In addition, 15 to 20% of the cells held at the restrictive temperature remained as single cells, and consistent with this, 15 to 20% of the cells held at the restrictive temperature had a 1N DNA content. Assuming that the nonbudded cells and the 1N DNA content cells are not simply dead cells, one interpretation of these latter results is that the rfa1-t6 and rfa1-t33 alleles also cause a defect in the initiation stage of DNA replication leading to some of the cells arresting in G1. These genetic properties of the rfa1-t6 and rfa1-33 alleles are consistent with the view that RPA1 is an essential DNA replication factor both in the initiation and the elongation stages (TSURIMOTO and STILLMAN 1989 Down; KENNY et al. 1990 Down; COLLINS and KELLY 1991 Down; LEE et al. 1991 Down; DORNREITER et al. 1992 Down).

A considerable amount of data is now available about the structure and function of different regions of RFA1, allowing us to relate the phenotypes of the different rfa1 mutations described here to the functional regions of RFA1 in which they map (Figure 8 and Figure 9). Five mutations, including the rfa1-t11 allele that caused strong defects in HO-induced double-strand break repair and SSA recombination, mapped in the region of RFA1 required for pol {alpha} stimulation (KIM et al. 1996 Down), a region whose in vivo function is not known. These mutants generally have strong UV S, MMSS, and HOS phenotypes, indicating this region is important for recombination and repair. One of these mutants was also weakly ts, suggesting this region might not be very important for DNA replication. Three mutations mapped in the region between the regions required for pol {alpha} stimulation and DNA binding, and all of these caused very weak phenotypes suggesting this region is also of limited functional significance or is tolerant of the amino acid changes observed. These results are consistent with the observation that this region of the protein is not required for SV40 replication in vitro (GOMES and WOLD 1996 Down). Mutations were found in both domains 1 and 2 of the ssDNA-binding region (GOMES and WOLD 1995 Down; KIM et al. 1996 Down; LIN et al. 1996 Down; BOCHKAREV et al. 1997 Down). Mutations in the N-terminal half of each of the two domains caused strong UV S and MMSS phenotypes, whereas mutations in the C-terminal half of domain 2 only caused weak UV S and MMSS phenotypes. Interestingly, none of the mutations in domain 1 caused a ts phenotype, whereas many of the domain 2 mutations caused a ts phenotype. Of particular significance in this regard were mutant alleles rfa1-m51 and rfa1-t6, which each caused a Ser-to-Pro change at the equivalent amino acid in domains 1 and 2, respectively; yet rfa1-m51 caused a strong UV S, MMSS phenotype whereas rfa1-t6 caused a strong ts phenotype in combination with a weak UV S, MMSS phenotype that was considerably weaker than the UV S, MMSS phenotype caused by the rfa1-m51 mutation. These data suggest that domains 1 and 2 do not play equivalent roles in RFA1. In some regards, mutations in domain 1 are reminiscent of the E. coli ssb113 mutation that alters the DNA-binding properties on SSB in a way that affects recombination and repair but not DNA replication (MEYER and LAINE 1990 Down). Three mutations were found in the Zn finger domain that is in close proximity to the region required for RFA holocomplex formation, and therefore interaction between RFA1 and RAD52 via RFA2 while an additional mutation mapped in the holocomplex formation region (ERDILE et al. 1991 Down; ADACHI and LAEMMLI 1992 Down; BROWN et al. 1994 Down; GOMES and WOLD 1996 Down; ISHIAI et al. 1996 Down; LIN et al. 1996 Down; LIN et al. 1997). Interestingly, three of these mutations caused the greatest sensitivity to HO endonuclease-induced double-strand breaks. These mutations caused strong UV S, MMSS, and HOS phenotypes but did not cause a ts phenotype. It is possible that these phenotypes result from an altered ability of mutant RFA1 proteins to recruit proteins like RAD52 required for recombination and repair (PARK et al. 1996 Down) because of alterations in holocomplex structure and/or RFA1-RFA2 interactions. The detailed biochemical analysis of these mutant proteins may prove insightful in understanding the biochemical functions of RPA.

In summary, we have described the isolation of 24 rfa1 mutants having a variety of different ts, UV S, and MMSS phenotypes. The analysis of these mutants has yielded at least two significant results not previously obtained in studies of rfa1 mutants. First, we have described the first strong double-strand break repair-defective and SSA-defective rfa1 mutant, providing strong evidence for a role of RPA in different types of double-strand break repair. Second, we have described a variety of ts lethal rfa1 mutations, and our data suggest that at least two of these mutations confer cell cycle progression defects consistent with RPA having roles in both the initiation and elongation stages of DNA replication. Third, we have provided genetic evidence that the replicative and recombination/repair properties of RPA may be separable. Continued analysis of the mutant collection described here is likely to provide insight into other roles that RPA may have in DNA metabolism.


*  FOOTNOTES

1 Present address: Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), Nara 630-01, JAPAN. Back
2 Present address: Ludwig Institute for Cancer Research, UC San Diego School of Medicine, La Jolla, CA 92093-0660. Back
3 Replication Protein A has been given several different names. In this article, the protein and protein subunits are called RPA as this is the first name we associate with the protein. Genes and mutant alleles are called RFA and rfa, respectively, as this is the official designation in Saccharomyces cerevisiae. Back


*  ACKNOWLEDGMENTS

We are grateful to TATSUYA MAEDA for helpful technical advice and discussions from the beginning of this work. We are indebted to JAMES LIPFORD and the Molecular Biology Core Facility, Dana-Farber Cancer Institute, for sequence analysis of rfa1 mutations; to CARLENE MCLEAN for technical assistance; to BRUCE STILLMAN and TATSUYA MAEDA for providing the YSB89 strain and the pRS series plasmids, respectively. This work was supported by National Institutes of Health grants GM-50006 (R.D.K.) and GM-20056 (J.E.H.).

Manuscript received June 13, 1997; Accepted for publication December 2, 1997.


*  LITERATURE CITED
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
 *RESULTS
 *DISCUSSION
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Function of a Conserved Checkpoint Recruitment Domain in ATRIP Proteins
Mol. Cell. Biol., May 1, 2007; 27(9): 3367 - 3377.
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 Nucleic Acids ResHome page
N. Grandin and M. Charbonneau
Control of the yeast telomeric senescence survival pathways of recombination by the Mec1 and Mec3 DNA damage sensors and RPA
Nucleic Acids Res., February 16, 2007; 35(3): 822 - 838.
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 Mol. Cell. Biol.Home page
K. Herzberg, V. I. Bashkirov, M. Rolfsmeier, E. Haghnazari, W. H. McDonald, S. Anderson, E. V. Bashkirova, J. R. Yates III, and W.-D. Heyer
Phosphorylation of Rad55 on Serines 2, 8, and 14 Is Required for Efficient Homologous Recombination in the Recovery of Stalled Replication Forks
Mol. Cell. Biol., November 15, 2006; 26(22): 8396 - 8409.
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 J. Biol. Chem.Home page
J. Majka, S. K. Binz, M. S. Wold, and P. M. J. Burgers
Replication Protein A Directs Loading of the DNA Damage Checkpoint Clamp to 5'-DNA Junctions
J. Biol. Chem., September 22, 2006; 281(38): 27855 - 27861.
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 J. Biol. Chem.Home page
S. Guo, Y. Zhang, F. Yuan, Y. Gao, L. Gu, I. Wong, and G.-M. Li
Regulation of Replication Protein A Functions in DNA Mismatch Repair by Phosphorylation
J. Biol. Chem., August 4, 2006; 281(31): 21607 - 21616.
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 Proc. Natl. Acad. Sci. USAHome page
S. Banerjee, S. Smith, and K. Myung
Suppression of gross chromosomal rearrangements by yKu70-yKu80 heterodimer through DNA damage checkpoints
PNAS, February 7, 2006; 103(6): 1816 - 1821.
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 J. Biol. Chem.Home page
S.-M. Kim, A. Kumagai, J. Lee, and W. G. Dunphy
Phosphorylation of Chk1 by ATM- and Rad3-related (ATR) in Xenopus Egg Extracts Requires Binding of ATRIP to ATR but Not the Stable DNA-binding or Coiled-coil Domains of ATRIP
J. Biol. Chem., November 18, 2005; 280(46): 38355 - 38364.
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 Nucleic Acids ResHome page
C.-J. Park, J.-H. Lee, and B.-S. Choi
Solution structure of the DNA-binding domain of RPA from Saccharomyces cerevisiae and its interaction with single-stranded DNA and SV40 T antigen
Nucleic Acids Res., July 25, 2005; 33(13): 4172 - 4181.
[Abstract] [Full Text] [PDF]

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 Mol. Cell. Biol.Home page
M. Clerici, V. Baldo, D. Mantiero, F. Lottersberger, G. Lucchini, and M. P. Longhese
A Tel1/MRX-Dependent Checkpoint Inhibits the Metaphase-to-Anaphase Transition after UV Irradiation in the Absence of Mec1
Mol. Cell. Biol., December 1, 2004; 24(23): 10126 - 10144.
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 Mol. Cell. Biol.Home page
D. Nakada, Y. Hirano, and K. Sugimoto
Requirement of the Mre11 Complex and Exonuclease 1 for Activation of the Mec1 Signaling Pathway
Mol. Cell. Biol., November 15, 2004; 24(22): 10016 - 10025.
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 Proc. Natl. Acad. Sci. USAHome page
K. Myung, S. Smith, and R. D. Kolodner
Mitotic checkpoint function in the formation of gross chromosomal rearrangements in Saccharomyces cerevisiae
PNAS, November 9, 2004; 101(45): 15980 - 15985.
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 Plant Physiol.Home page
A. L. Contento, S.-J. Kim, and D. C. Bassham
Transcriptome Profiling of the Response of Arabidopsis Suspension Culture Cells to Suc Starvation
Plant Physiology, August 1, 2004; 135(4): 2330 - 2347.
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 J. Bacteriol.Home page
C. Lindner, R. Nijland, M. van Hartskamp, S. Bron, L. W. Hamoen, and O. P. Kuipers
Differential Expression of Two Paralogous Genes of Bacillus subtilis Encoding Single-Stranded DNA Binding Protein
J. Bacteriol., February 15, 2004; 186(4): 1097 - 1105.
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 Nucleic Acids ResHome page
Y. Ono, K. Tomita, A. Matsuura, T. Nakagawa, H. Masukata, M. Uritani, T. Ushimaru, and M. Ueno
A novel allele of fission yeast rad11 that causes defects in DNA repair and telomere length regulation
Nucleic Acids Res., December 15, 2003; 31(24): 7141 - 7149.
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 Mol. Cell. Biol.Home page
S. E. Lee, A. Pellicioli, M. B. Vaze, N. Sugawara, A. Malkova, M. Foiani, and J. E. Haber
Yeast Rad52 and Rad51 Recombination Proteins Define a Second Pathway of DNA Damage Assessment in Response to a Single Double-Strand Break
Mol. Cell. Biol., December 1, 2003; 23(23): 8913 - 8923.
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 Proc. Natl. Acad. Sci. USAHome page
L. Zou, D. Liu, and S. J. Elledge
Replication protein A-mediated recruitment and activation of Rad17 complexes
PNAS, November 25, 2003; 100(24): 13827 - 13832.
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 J. Biol. Chem.Home page
S. K. Binz, Y. Lao, D. F. Lowry, and M. S. Wold
The Phosphorylation Domain of the 32-kDa Subunit of Replication Protein A (RPA) Modulates RPA-DNA Interactions: EVIDENCE FOR AN INTERSUBUNIT INTERACTION
J. Biol. Chem., September 12, 2003; 278(37): 35584 - 35591.
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 J. Biol. Chem.Home page
N. Kantake, T. Sugiyama, R. D. Kolodner, and S. C. Kowalczykowski
The Recombination-deficient Mutant RPA (rfa1-t11) Is Displaced Slowly from Single-stranded DNA by Rad51 Protein
J. Biol. Chem., June 20, 2003; 278(26): 23410 - 23417.
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 Nucleic Acids ResHome page
K.-H. Bae, H.-S. Kim, S.-H. Bae, H.-Y. Kang, S. Brill, and Y.-S. Seo
Bimodal interaction between replication-protein A and Dna2 is critical for Dna2 function both in vivo and in vitro
Nucleic Acids Res., June 15, 2003; 31(12): 3006 - 3015.
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 ScienceHome page
L. Zou and S. J. Elledge
Sensing DNA Damage Through ATRIP Recognition of RPA-ssDNA Complexes
Science, June 6, 2003; 300(5625): 1542 - 1548.
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 Microbiol. Mol. Biol. Rev.Home page
L. S. Symington
Role of RAD52 Epistasis Group Genes in Homologous Recombination and Double-Strand Break Repair
Microbiol. Mol. Biol. Rev., December 1, 2002; 66(4): 630 - 670.
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A. L. Eggler, R. B. Inman, and M. M. Cox
The Rad51-dependent Pairing of Long DNA Substrates Is Stabilized by Replication Protein A
J. Biol. Chem., October 11, 2002; 277(42): 39280 - 39288.
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 Mol. Cell. Biol.Home page
P. J. Lau, H. Flores-Rozas, and R. D. Kolodner
Isolation and Characterization of New Proliferating Cell Nuclear Antigen (POL30) Mutator Mutants That Are Defective in DNA Mismatch Repair
Mol. Cell. Biol., October 1, 2002; 22(19): 6669 - 6680.
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 GeneticsHome page
C. Soustelle, M. Vedel, R. Kolodner, and A. Nicolas
Replication Protein A Is Required for Meiotic Recombination in Saccharomyces cerevisiae
Genetics, June 1, 2002; 161(2): 535 - 547.
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 Mol. Cell. Biol.Home page
C. Ramilo, L. Gu, S. Guo, X. Zhang, S. M. Patrick, J. J. Turchi, and G.-M. Li
Partial Reconstitution of Human DNA Mismatch Repair In Vitro: Characterization of the Role of Human Replication Protein A
Mol. Cell. Biol., April 1, 2002; 22(7): 2037 - 2046.
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 J. Cell Sci.Home page
P. Grandi, M. Eltsov, I. Nielsen, and I. Raska
DNA double-strand breaks induce formation of RP-A/Ku foci on in vitro reconstituted Xenopus sperm nuclei
J. Cell Sci., March 11, 2002; 114(18): 3345 - 3357.
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 GeneticsHome page
A. P. Davis and L. S. Symington
The Yeast Recombinational Repair Protein Rad59 Interacts With Rad52 and Stimulates Single-Strand Annealing
Genetics, October 1, 2001; 159(2): 515 - 525.
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 Proc. Natl. Acad. Sci. USAHome page
H. Debrauwere, S. Loeillet, W. Lin, J. Lopes, and A. Nicolas
Links between replication and recombination in Saccharomyces cerevisiae: A hypersensitive requirement for homologous recombination in the absence of Rad27 activity
PNAS, July 17, 2001; 98(15): 8263 - 8269.
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 Mol. Cell. Biol.Home page
V. Paciotti, M. Clerici, M. Scotti, G. Lucchini, and M. P. Longhese
Characterization of mec1 Kinase-Deficient Mutants and of New Hypomorphic mec1 Alleles Impairing Subsets of the DNA Damage Response Pathway
Mol. Cell. Biol., June 15, 2001; 21(12): 3913 - 3925.
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 Mol. Cell. Biol.Home page
H.-S. Kim and S. J. Brill
Rfc4 Interacts with Rpal and Is Required for Both DNA Replication and DNA Damage Checkpoints in Saccharomyces cerevisiae
Mol. Cell. Biol., June 1, 2001; 21(11): 3725 - 3737.
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 Mol. Cell. Biol.Home page
L. E. Kang and L. S. Symington
Aberrant Double-Strand Break Repair in rad51 Mutants of Saccharomyces cerevisiae
Mol. Cell. Biol., December 15, 2000; 20(24): 9162 - 9172.
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 GeneticsHome page
D. J. Tomso and K. N. Kreuzer
Double-Strand Break Repair in Tandem Repeats During Bacteriophage T4 Infection
Genetics, August 1, 2000; 155(4): 1493 - 1504.
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 Mol. Cell. Biol.Home page
L. Zou and B. Stillman
Assembly of a Complex Containing Cdc45p, Replication Protein A, and Mcm2p at Replication Origins Controlled by S-Phase Cyclin-Dependent Kinases and Cdc7p-Dbf4p Kinase
Mol. Cell. Biol., May 1, 2000; 20(9): 3086 - 3096.
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 Microbiol. Mol. Biol. Rev.Home page
F. Paques and J. E. Haber
Multiple Pathways of Recombination Induced by Double-Strand Breaks in Saccharomyces cerevisiae
Microbiol. Mol. Biol. Rev., June 1, 1999; 63(2): 349 - 404.
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 GeneticsHome page
M. P. Colaiácovo, F. Pâques, and J. E. Haber
Removal of One Nonhomologous DNA End During Gene Conversion by a RAD1- and MSH2-Independent Pathway
Genetics, April 1, 1999; 151(4): 1409 - 1423.
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 Mol. Cell. Biol.Home page
S. J. Brill and S. Bastin-Shanower
Identification and Characterization of the Fourth Single-Stranded-DNA Binding Domain of Replication Protein A
Mol. Cell. Biol., December 1, 1998; 18(12): 7225 - 7234.
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 J. Biol. Chem.Home page
M. S. DeMott, S. Zigman, and R. A. Bambara
Replication Protein A Stimulates Long Patch DNA Base Excision Repair
J. Biol. Chem., October 16, 1998; 273(42): 27492 - 27498.
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 J. Biol. Chem.Home page
T. A. Ranalli, M. S. DeMott, and R. A. Bambara
Mechanism Underlying Replication Protein A Stimulation of DNA Ligase I
J. Biol. Chem., January 11, 2002; 277(3): 1719 - 1727.
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