Functional Analysis of Human Replication Protein A in Nucleotide Excision Repair*

Human replication protein A (RPA) is a three-subunit protein complex (70-, 34-, and 11-kDa subunits) involved in DNA replication, repair, and recombination. Both the 70- (p70) and 34-kDa (p34) subunits interact with Xeroderma pigmentosumgroup A complementing protein (XPA), a key protein involved in nucleotide excision repair. Our deletion analysis indicated that no particular domain(s) of RPA p70 was essential for its interaction with XPA, whereas 33 amino acids from the C terminus of p34 (p34Δ33C) were necessary for the XPA interaction. Furthermore, mutant RPA lacking the p34 C terminus failed to interact with XPA, suggesting that p34, not p70, is primarily responsible for the interaction of RPA with XPA. RPA stimulated the interaction of XPA with UV-damaged DNA through an RPA-XPA complex on damaged DNA sites because (i) the RPA mutant lacking the C terminus of p34 failed to stimulate an XPA-DNA interaction, and (ii) the ssDNA binding domain of RPA (amino acids 296–458) was necessary for the stimulation of the XPA-DNA interaction. Two separate domains of p70, a single-stranded DNA binding domain and a zinc-finger domain, were necessary for RPA function in nucleotide excision repair. The mutant RPA (RPA:p34Δ33C), which lacks its stimulatory effect on the XPA-DNA interaction, also poorly supported nucleotide excision repair, suggesting that the XPA-RPA interaction on damaged DNA is necessary for DNA repair activity.

Human replication protein A (also known as human singlestranded DNA-binding protein) is composed of three tightly associated polypeptides (70-, 34-, and 11-kDa subunits). It was originally identified as a factor required for in vitro SV40 DNA replication (1)(2)(3). In SV40 DNA replication, RPA 1 interacts with SV40 Tag and the DNA polymerase ␣-primase complex at the origin, where it participates in the initiation process (4 -8). During replication, RPA binds to and stabilizes the ssDNA generated at the replication forks and stimulates DNA polymerase ␣ activity, suggesting it has a role in both initiation and elongation (9 -11). Recent mutational analysis, however, indicates that the ssDNA binding activity of RPA, but not its ability to stimulate DNA polymerase, is essential for SV40 DNA replication (12).
The involvement of RPA in DNA repair has been shown in human and yeast nucleotide excision repair systems (13)(14)(15)(16). Similar to its function in replication, RPA appears to be involved in multiple stages of the DNA repair process. Its role in the early stage of repair was suggested because the RPA requirement can be bypassed by the presence of Escherichia coli UvrABC, which incises damaged DNA (14). This early stage involvement includes the interaction of RPA with the UV damage recognition factor XPA (17)(18)(19)(20). After forming an RPA-XPA complex on damaged DNA, RPA recruits XPG and ERCC1-XPF, two endonucleases that are likely involved in the 3Ј and 5Ј incision reactions (18,21) to the damaged site. RPA is also involved in the later stage gap-filling reaction, which requires PCNA, RF-C (A1), and DNA polymerase ␦ (or ⑀) (22). In the mammalian system, RPA p34 phosphorylation is induced by DNA damage (UV or x-ray). DNA-dependent protein kinase (DNA-PK) was shown to be responsible for the hyperphosphorylation of RPA p34 (23,24); however, recent in vivo observations suggest the possible involvement of other kinases (25). Human and mouse cells deficient in DNA-PK activity are not only defective in site-specific recombination but are also very sensitive to DNA damage, suggesting a role for DNA-PK (and possibly RPA phosphorylation) in these processes (26,27). In addition, extracts from UV-irradiated HeLa cells poorly support SV40 DNA replication compared with those from nonirradiated cells (23). UV damage-induced inhibition of replication correlates with RPA phosphorylation and is reversed by the addition of purified RPA to an in vitro system, suggesting a role for RPA phosphorylation in damage-induced inhibition of replication (23). However, no biologic function for RPA phosphorylation has yet been identified.
RPA is involved in recombination through its stimulation of strand exchange reactions in vitro (28) and its requirement in homologous recombination (29). Recently, RPA was shown to interact with Rad52, a protein required for recombination and strand-break repair (30,31). In addition, the RPA-Rad52 interaction seems to be required for homologous recombination in vivo (32).
RPA forms a complex in an orderly manner such that p34 and p11 form a stable subcomplex to which p70 is then added (33,34). The p70 subunit has multiple functional domains, an N-terminal domain for interacting with pol ␣ (12, 35), p53 (36), two middle subdomains with DNA binding activity (12, 37), four cysteine-type zinc finger domains (amino acids 481-503), and a C-terminal domain for interacting with other RPA subunits (12,37). The p34 phosphorylation sites for DNA-PK and cyclin-dependent kinase have recently been mapped to its Nterminal domain (38,39).
In this report, we analyzed RPA function in nucleotide excision repair by examining various RPA mutants. We found that the C-terminal domain of RPA p34 is responsible for RPA interaction with XPA. This interaction is necessary for the formation of a stable RPA-XPA complex on DNA. An RPA mutant lacking the p34 C terminus poorly supported DNA repair, which strongly suggests that the XPA-RPA interaction is necessary for efficient DNA repair. We also found two p70 domains (a ssDNA binding domain and a zinc finger motif) that were essential for nucleotide excision repair.
The DNA plasmid, p5A (4.5 kbp), represents pBluescript (pBS, 3 kbp) in which the 1.45-kb full-length cDNA for RPA p34 had been inserted. This plasmid was prepared for the nucleotide excision repair assay by two rounds of CsCl gradient centrifugation (40) followed by 5-20% sucrose density gradient centrifugation at 25,000 rpm for 19 h at 2°C. pBS (3 kbp) was prepared by two rounds of CsCl centrifugation from the E. coli strain JM109 (recA), without chloramphenicol amplification. The purified pBS (500 g) was suspended in 10 ml of TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) and thin-layered in a plastic Petri dish (150 mm) with gentle rocking. The Petri dish was then UV-irradiated with germicidal UV light (G15T8) at a fluence rate of 0.9 watt/m 2 . UV fluence was measured by using a Black-Ray UV meter (UVP, Inc., Upland, CA). After UV irradiation, the DNA was treated with E. coli Nth protein, and closed circular supercoiled DNA was isolated by CsCl gradient centrifugation followed by sucrose gradient centrifugation, as described previously (41). Even though the primary source of UV was UV-C (254 nm in wavelength), the final preparation of irradiated plasmid DNA contained more than 90% supercoiled circular duplex DNA (data not shown).
Metabolic Labeling of Sf-9 Cells with [ 35 S]Methionine and Immunoprecipitation-[ 35 S]Methionine-labeled cell lysates were prepared as described previously (33). Sf-9 cells (2.0 ϫ 10 6 ) were plated on a 60-mm dish and infected with individual recombinant baculoviruses encoding RPA subunits at a multiplicity of infection of 15 for approximately 40 h at 27°C. The insect cells were then labeled with trans 35 S-labeled methionine at 200 Ci/ml (1200 -1600 Ci/mmol) for 4 h in methionine-free medium containing 5% dialyzed fetal calf serum. Cells were briefly washed with phosphate-buffered saline and lysed for 1 h on ice in 0.5 ml of lysis buffer (50 mM Tris-HCl, pH 8.0, 250 mM NaCl, 0.5% Nonidet P-40, 1 mM DTT, 1 mM EDTA, 0.1 mM NaF, 10 mM ␤-glycerophosphate, 0.1 mM sodium orthovanadate, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mg/ml leupeptin, and 0.2 mg/ml antipain). For immunoprecipitation, cleared cell lysates (50 l) were incubated with the indicated polyclonal antibody (5 l) in the presence of bovine serum albumin (200 g/ml) at 4°C with rocking. Protein A-Sepharose was then added, and the mixture was incubated for 1 h at 4°C. Immunoprecipitates were collected by centrifugation, washed 5 times with cell lysis buffer, and analyzed by 12% SDS-polyacrylamide gel electrophoresis (PAGE).
GST Fusion Pull-down Assay-Unless otherwise specified, aliquots (0.4 -1.0 ml) of GST fusion protein containing lysates were adjusted to 0.5% nonfat dried milk and add to 25 l of glutathione-Sepharose beads that had been washed three times and resuspended (1:1 (v/v)) in NETN buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40) containing 0.5% dried milk. The aliquots were then rocked for 30 min at 4°C. After being washed 3 times with NETN containing 0.5% dried milk, the beads were incubated with lysates containing target protein (or purified protein) for 1 h at 4°C on a rocker and then washed 5 times with NETN. For a direct analysis of the proteins bound to the beads, the beads were mixed with SDS-PAGE sample buffer, and proteins were resolved on SDS-PAGE. Proteins were then visualized either by fluorography or immunoblotting. To purify bound GST-XPA, proteins bound to the GST beads were eluted with elution buffer (40% ethylene glycol, 10% glycerol, 1.5 M NaCl, 0.01% Nonidet P-40, 1 mM EDTA, and 1 mM DTT), and dialyzed against buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.02% Nonidet P-40, 1 mM EDTA, 1 mM DTT, 10% glycerol, 0.1 mM phenylmethylsulfonyl fluoride, 100 g/l antipain and leupeptin) overnight.
Preparation of Proteins-Wild-type RPA and RPA mutants, PCNA, and XPA were isolated by the procedures described previously (12,20). The E. coli endonuclease III-overproducing strain was originally obtained from Dr. J. Cunningham (State University of New York, Albany, NY) and purified according to the published procedure (43). DNA-PK was kindly provided by Dr. C. Anderson (Brookhaven National Laboratory, Long Island, NY) (44).
To prepare protein fractions lacking RPA, HeLa cell extracts (20 ml, 350 mg of protein) were ammonium sulfate-fractionated as described previously (1). The ammonium sulfate-(35-65%) fraction was dialyzed against buffer A containing 150 mM KCl and loaded onto a phosphocellulose column (PC; 15 ml) that was preequilibrated with buffer A containing 150 mM KCl. After washing the column with the same buffer, bound proteins were eluted with buffer A containing 1.0 M KCl. The PC-1.0 M fraction, which represents the fraction of HeLa whole cell extracts that bound to the PC column in the presence of 0.1 M KCl and is eluted with 1.0 M KCl, was pooled and dialyzed against buffer A (25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM DTT, 0.01% Nonidet P-40, and 10% glycerol) containing 50 mM KCl and then used as a receptor fraction for the RPA complementation assay in repair (the PC-1.0 M fraction also lacks the repair protein PCNA).
The RPA-XPA Interaction on DNA-To prepare the DNA substrate, pBS plasmid was digested with BstN1 and a 121-bp fragment isolated from the agarose gel. After being labeled with [␥-32 P]ATP by the action of polynucleotide kinase, the DNA was irradiated with a germicidal UV lamp to prepare UV-damaged DNA. The reaction mixtures (100 l) contained 25 mM Hepes-KOH (pH 7.5), 5 mM MgCl 2 , 1 mM DTT, 100 mM KCl, bovine serum albumin (200 g/ml), 0.1 g of poly(dI:dC), 50 fmol of either irradiated or nonirradiated 5Ј-[ 32 P]DNA (121 bp), and the indicated amounts of GST-XPA and/or RPA. After a 15-min incubation at 30°C, NETN buffer containing 0.5% dried milk and GST beads was added, and the reactions were processed as described for the GST fusion pull-down assay. After the GST beads were washed, the samples were analyzed for [ 32 P]DNA on a 5% polyacrylamide gel containing 0.1% SDS.
Nucleotide Excision Repair in Vitro-Nucleotide excision repair of UV-damaged DNA was carried out according to the published procedure (41). Reaction mixtures (50 l) contained 0.2 g each of UVirradiated (450 J/m 2 ) pBS (3 kbp) and nonirradiated p5A (4.5 kbp), 40 mM creatine phosphate-di-Tris salt, pH 7.7, 1 g of creatine kinase, 50 mM Hepes-KOH, pH 7.8, 70 mM KCl, 7.5 mM MgCl 2 , 0.5 mM DTT, 0.4 mM EDTA, 2 mM ATP, 20 M each dGTP, dCTP, TTP, 8 M [␣-32 P]dATP (25,000 cpm/pmol), 5 g of bovine serum albumin, and 150 g of the PC-1.0 M fraction of HeLa whole cell extracts. After incubation for 3 h at 30°C, DNA was isolated from the reaction mixtures, linearized with BamHI, and separated by 1% agarose gel electrophoresis in the presence of 0.5 g/ml ethidium bromide. The DNA and repair products were analyzed by both fluorography and exposure to x-ray film.

RESULTS
The C Terminus of p34 Is Primarily Responsible for Interaction of RPA with XPA-RPA interacts with SV40 Tag, XPA, and human Rad52, key proteins involved in replication, repair, and recombination, respectively. These interactions are essential to the function of RPA in DNA metabolism. We and others have shown that both p70 and p34 interact with XPA, Rad52 (17,18,32), and SV40 Tag (data not shown) and have suggested that RPA may function in all three DNA metabolic events by interacting with key proteins in replication, repair, and recombination.
To identify the domain(s) of RPA p70 and p34 involved in these protein-protein interactions, we examined p70 and p34 deletion mutants for their ability to interact with XPA. An GST-XPA fusion protein was used to pull down the metabolically labeled RPA p70 or p34 (Fig. 1, panel A). Compared with the amount of mutant p70 immunoprecipitated by the anti-p70 antibody, only a fraction of the p70 in lysates was pulled down by the GST fusion protein (Fig. 1, panel B). In addition, no particular domain for p70 binding to XPA was identified. In contrast, p34 interacted efficiently with XPA (Fig. 1, panel C). Of interest, XPA interacted with a p34 mutant that had an N-terminal deletion (p34⌬2-30) as well as a mutant that had a serine-to-alanine substitution in its cdk consensus sites (amino acids 23 and 29); however, XPA did not interact with a p34 mutant that lacked 33 amino acids at its C terminus (Fig. 1,  panel C).
To further analyze the RPA-XPA interaction, we examined purified RPA complexes containing either p34 that lacked the C terminus (p34⌬33C) or p70 that lacked the ssDNA binding domain (⌬RS) for their interaction with XPA. Mutant RPA that lacked the p34 C terminus failed to interact with XPA, whereas RPA containing the p70 mutant (⌬RS) interacted with XPA (Fig. 2). All other RPAs containing various p70 mutants also supported the RPA-XPA interaction (data not shown). It should be pointed out, however, that our failure to detect p34⌬33C in the GST-XPA pulldown assay (Fig. 2B) might be due to the much reduced band intensities compared with the Western blot band intensities shown in Fig. 2A. Overall, our result suggests that XPA interacts with RPA primarily through the C terminus of p34.
RPA-XPA Interaction on UV-damaged DNA-Previous reports demonstrated that XPA binding to damaged DNA is enhanced by the presence of RPA (18,19), even though both XPA and RPA can independently bind to damaged DNA (45,46). We examined the effect of RPA on the interaction of XPA with DNA by using purified GST-XPA in a pull-down assay to measure the interaction of XPA with a 5Ј-labeled 121-bp fragment of damaged and nondamaged dsDNA. The purified GST-XPA used in this experiment was described previously (17) and was functionally active in nucleotide excision repair (data not shown). Consistent with a previous study (45), XPA alone showed a strong interaction with damaged DNA (8 kJ/m 2 ) but not with nondamaged DNA (Fig. 3, panel A). The XPA-damaged DNA interaction was significantly stimulated (up to 4-fold) by the addition of RPA (Fig. 3, panel B). The weak interaction between XPA and nondamaged DNA was also stimulated by RPA (Fig. 3, panel B, lanes 2-4), which may be explained by the fact that RPA binds to dsDNA in a nonspecific manner (9, 11). We examined whether the stimulatory effect of RPA on the XPA-DNA interaction occurs through the interaction of RPA with XPA on DNA. For this, we compared the XPA-DNA interaction of wild-type RPA with that of mutants lacking either the N-terminal or C-terminal domain of p34 (RPA:p34⌬2-30 and RPA:p34⌬33C, respectively) (Fig. 4). The wild-type RPA and RPA:p34⌬2-30 showed similar stimulatory effects on XPA-DNA, whereas RPA:p34⌬33C had virtually no effect. We also examined the effects of mutant RPAs on the XPA-damaged DNA interaction. As shown in Fig. 5, mutant RPAs lacking the N-terminal domain (RPAp70⌬2-100 and RPAp70JK (⌬101-169)) showed a 25-50% reduction in their ability to stimulate the XPA-DNA interaction, whereas mutant RPAs lacking the ssDNA binding domain (RPAp70PQ and RPAp70RS; see Ref. 12 for details) were totally inactive. Our results strongly indicate that RPA forms a complex with XPA on damaged DNA and thereby stabilizes the interaction of XPA with the DNA.
DNA damage induces phosphorylation of RPA and inhibits DNA replication in S-phase cells (23,24,47), suggesting a possible role for RPA phosphorylation in repair and replication. However, both phosphorylated and nonphosphorylated RPA efficiently interacted with XPA, indicating that the interaction of RPA with XPA does not require phosphorylation of RPA and can occur in the absence of DNA (Fig. 6).
The C-terminal Domain of p34 Is Necessary for the Function of RPA in Repair-Because the C-terminal domain of RPA p34 is responsible for the interaction of RPA with XPA (Figs. 2 and  3), we examined whether this interaction was also necessary for the function of RPA in DNA repair. It has previously been suggested that the RPA-XPA interaction is required for DNA repair (19). We, therefore, examined the ability of RPAs containing different p34 mutants to support nucleotide excision repair activity. The RPAp34⌬33C mutant poorly supported nucleotide excision repair in vitro, whereas an RPA mutant lacking phosphorylation sites (⌬2-30) efficiently supported repair activity (Fig. 7). This result is in keeping with our results on the stimulation of the interaction between XPA and damaged DNA and suggests that the interaction of RPAp34 with XPA is necessary for nucleotide excision repair. These results also support the previous observation that RPA phosphorylation is not required for nucleotide excision repair (48).
The ssDNA Binding Activity of RPA Is Required for Nucleotide Excision Repair-RPA p70 contains two large DNA binding domains, an N-terminal domain for its interaction with the polymerase ␣-primase complex, and a C-terminal domain for its interaction with the two other RPA subunits (12,(35)(36)(37). We examined various RPAs containing p70 deletion mutants for their ability to support nucleotide excision repair. As shown in  5, 8, and 9) of wild-type (wt) RPA was added. The mixtures were processed as described in Fig. 3A and analyzed by 5% PAGE. For quantitation, bands corresponding to a 121-bp fragment were excised from the gel and measured for radioactivity by liquid scintillation counter.  2-4,  6, 7, 9, and 10) was added together with 20 ng (lanes 3, 6, and 9) or 100 ng (lanes 4, 5, 7, 8, 10, and 11) of either wild-type or mutant RPA. All other conditions were the same as those described in the legend to Fig. 3.  Fig. 8, the DNA binding domains of p70 were essential for DNA repair activity, but the N-terminal polymerase ␣ stimulation domain was not. Of interest, mutant RPA that lacked a zinc finger domain of p70 (RPAp70⌬TU) was unable to support nucleotide excision repair, suggesting a role for the zinc finger domain in this activity. In contrast, zinc finger domain deletion and substitution mutants efficiently support SV40 DNA replication in vitro (12). 2

DISCUSSION
RPA plays an essential role in multistaged nucleotide excision repair. To better understand the roles of the individual RPA subunits in repair, we analyzed a series of RPA mutants for their ability to repair DNA and to interact with XPA.
As reported previously (18), both the p70 and the p34 subunit of RPA interacted with XPA. Our GST fusion pull-down assay demonstrated that p70 interacted weakly with XPA and that no particular p70 domain was involved in this interaction. On the other hand, the interaction of p34 with XPA was very strong and required the C-terminal domain of p34, suggesting that the p34 C terminus is primarily responsible for the RPA-XPA interaction. In studies by others (19), however, deletion at conserved motifs in XPA abolished binding to p70 but not binding to p34. It is still possible that other p70 deletions or mutations that were not used in this study would cause loss of binding to XPA. Nonetheless, the C-terminal domain of p34 is highly acidic and conserved among eukaryotes (49,50), suggesting that this domain has an essential role in the regulation of DNA metabolism. In fact, the C-terminal domain of p34 is also involved in the RPA-Rad52 interaction (data not shown).
The RPA-XPA interaction is likely involved in recognition of damaged DNA because the stimulatory effect of RPA on the DNA binding activity pf XPA requires the RPA-XPA interaction. Furthermore, the RPA mutant that lacks ssDNA binding domain was not able to stimulate the interaction between XPA and damaged DNA. These results indicate that both RPA and XPA likely interact with each other on the damaged DNA to form a stable complex. It is conceivable that, upon DNA damage, XPA first contacts the damaged DNA and then recruits RPA to the damaged site. Both RPA and XPA contain C 4 -type zinc finger motifs and likely undergo structural changes upon DNA binding. Once XPA and RPA form a stable complex on the DNA, they are thought to bring other repair proteins to the site of initiation of nucleotide excision repair.
It has been suggested that the interaction of XPA with RPA  3, 6, 9, 12, 15, and 18) or 100 ng (lanes 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, and 20) of either wild-type (wt) or mutant RPA were included. All other conditions were the same as those described for Fig. 3.   FIG. 6. RPA phosphorylation has no effect on the RPA-XPA interaction. Reaction mixtures (60 l) contained 7 mM MgCl 2 , bovine serum albumin (200 g/ml), 4 mM ATP, and 1.0 g of purified wild-type (wt) RPA. Where indicated, 1.0 g of purified DNA-PK or 0.2 g of x174 (ssc) was added. After a 1-h incubation at 30°C, the mixtures were directly analyzed by 12% SDS-PAGE and Western blot analysis (panel A) or incubated with glutathione-Sepharose beads loaded with GST-XPA before Western blot analysis (panel B). An anti-p34 polyclonal antibody (from rabbit) was used for immunoblotting. is essential for nucleotide excision repair (18,19). Our result that an RPA mutant lacking the ability to interact with XPA poorly supports nucleotide excision repair in vitro is consistent with these previous observations. Furthermore, an RPA mutant that lacks the RPA phosphorylation sites supported nucleotide excision repair (Fig. 7), suggesting that RPA phosphorylation is not necessary for DNA repair (48). Consistent with this finding, our results also show that RPA phosphorylation has no effect on the interaction between RPA and XPA (Fig. 6).
We previously reported that XPA interacts with RPA and interferes with RPA function in DNA replication (20). In this report, however, we have demonstrated that the RPA-XPA interaction is necessary for nucleotide excision repair. Therefore, it is possible that the RPA-XPA complex, once formed, is used for repair and not for replication, which suggests some mechanism exists to differentially regulate RPA activity upon DNA damage.
The ssDNA binding domain of RPA is localized in the middle of the p70 subunit and is essential for DNA replication (12). Mutant RPA that lacks ssDNA binding activity does not support nucleotide excision repair (Fig. 8), suggesting that the ssDNA binding domain of RPA is essential for its function in DNA metabolism. Both XPA and RPA need to contact the DNA to form a stable complex (Figs. 4 and 5), and it may be that a mutant that lacks ssDNA binding activity cannot form a complex with XPA on DNA (Fig. 5). Also, the ssDNA binding activity of RPA may be necessary for a later stage of repair, such as the gap-filling reaction. RPA could bind to and stabilize the short patches of ssDNA that are generated by the incision/ excision process, facilitating the loading of RF-C (activator 1) and PCNA, which in turn promote recognition of the 3Ј-ends by DNA polymerase ␦ (or ⑀) (22). Since RPA is known to interact with damaged DNA (46), its ssDNA binding activity may also be necessary for this interaction.
Interestingly, an RPA mutant lacking the zinc finger domain (RPA:p70⌬TU) poorly supported nucleotide excision repair ( Fig. 8) but efficiently supported SV40 DNA replication (12). This result suggests distinct roles for RPA in replication and repair. Lin et al. (36) recently reported that a zinc finger mutant of RPA does not support SV40 DNA replication (36); however, we have consistently observed that our zinc finger mutants (both deletion and substitution) efficiently support SV40 DNA replication (12). 2 The addition of increasing amounts of zinc finger mutant RPA inhibits DNA polymerase ␣ and ␦ activities on primed DNA templates (12). Similarly, the zinc finger mutant could affect DNA polymerase activity during the gap-filling reaction, thereby inhibiting repair. Despite being involved in repair, this zinc finger mutant may not have a significant impact on DNA polymerase activity at the replication fork. Another possibility is that the zinc finger mutant affects the ability of RPA to bind DNA, particularly damaged DNA (46). We observed that the zinc finger RPA mutant (cysteine-to-alanine substitution) had reduced ssDNA binding activity compared with wild-type RPA. 2 It is not clear, however, whether this reduction in ssDNA binding activity contributes to the inability of the mutant to support nucleotide excision repair. Further characterization will be necessary to clarify the role of zinc finger domain of RPA in repair.