Characterization of genetic interactions with RFA1: the role of RPA in DNA replication and telomere maintenance

Biochimie 82 (2000) 71−78 © 2000 Société française de biochimie et biologie moléculaire/Éditions scientifiques et médicales Elsevier SAS. All rights reserved. S0300908400001838/FLA

Characterization of genetic interactions with RFA1: the role of RPA in DNA replication and telomere maintenance Julianne Smith**, Hui Zou***, Rodney Rothstein* Department of Genetics & Development, Columbia University College of Physicians & Surgeons, 701 West 168th Street, New York, NY 10032-2704, USA (Received 4 October 1999; accepted 19 November 1999) Abstract — Replication protein A (RPA) is a heterotrimeric single-stranded DNA binding protein whose role in DNA replication, recombination and repair has been mainly elucidated through in vitro biochemical studies utilizing the mammalian complex. However, the identification of homologs of all three subunits in Saccharomyces cerevisiae offers the opportunity of examining the in vivo role of RPA. In our laboratory, we have previously isolated a missense allele of the RFA1 gene, encoding the p70 subunit of the RPA complex. Strains containing this mutant allele, rfa1-D228Y, display increased levels of direct-repeat recombination, decreased levels of heteroallelic recombination, UV sensitivity and a S-phase delay. In this study, we have characterized further the role of RPA by screening other replication and repair mutants for a synthetic lethal phenotype in combination with the rfa1-D228Y allele. Among the replication mutants examined, only one displayed a synthetic lethal phenotype, pol12-100, a conditional allele of the B subunit of pol α-primase. In addition, a delayed senescence phenotype was observed in rfa1-D228Y strains containing a null mutation of HDF1, the S. cerevisiae homolog of the 70 kDa subunit of Ku. Interestingly, a synergistic reduction in telomere length observed in the double mutants suggests that the shortening of telomeres may be the cause of the decreased viability in these strains. Furthermore, this result represents the first evidence of a role for RPA in telomere maintenance. © 2000 Société française de biochimie et biologie moléculaire/Éditions scientifiques et médicales Elsevier SAS replication protein A / DNA replication / DNA polymerase alpha / Ku protein / telomere maintenance

1. Introduction Replication protein A (RPA) is a single-stranded DNA binding protein (SSB) that is required for several processes in DNA metabolism, including replication, repair and recombination. Homologs have been identified in every eukaryotic organism examined and are all heterotrimeric proteins composed of subunits of approximately 70, 30 and 14 kDa (for review see [1]). Originally identified as a HeLa cell protein required for in vitro replication of simian virus 40 DNA [2-4], RPA was subsequently shown to play multiple roles in this process. In the initiation stage, RPA promotes T antigen-dependent unwinding of the replication origin and DNA polymerase α-primase activity [5-7]. There is a specific requirement for RPA in this step due to protein interactions with both SV40 T-antigen and the DNA polymerase α-primase complex [8]. RPA is also necessary for DNA elongation and has been shown to stimulate the activity of polymerase α, δ and e [9-11]. * Correspondence and reprints ** Present address: UMR 218, Institut Curie, 26, rue d’Ulm, 75248 Paris cedex 05, France *** Present address: Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA

Biochemical studies have also demonstrated a role for RPA in DNA repair and recombination processes. Nucleotide excision repair studies carried out with purified protein components have demonstrated an absolute requirement for RPA [12, 13]. The complex has been implicated in the recognition step due to its ability to preferentially bind UV-damaged DNA [14] and to interact with and enhance the damage recognition capabilities of the XPA protein [15]. Additionally, a role for RPA in the incision of damaged DNA is indicated by its ability to associate with the structure specific nucleases XPG and ERCC1/XPF [16]. In recombination, RPA has been shown to stimulate in vitro the pairing activity of Rad52 [17] as well as the strand-exchange activity of Rad51, the eukaryotic RecA homolog [18, 19]. A physical association has also been detected between RPA and the Rad51 and the Rad52 proteins [20-22] suggesting that specific protein interactions are required for its activity. The 70 kDa subunit represents the most extensively characterized component of the RPA complex. It is this subunit that is involved in DNA-binding [23] and is implicated in the majority of the described protein interactions [1]. Biochemical studies have delineated three distinct domains of the protein. The N-terminal domain (aa 1–170) that is involved in RPA-protein interactions, a central DNA binding domain (aa 170–450) and a

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Table I. S. cerevisiae strains used in this study. Straina

Genotype

Origin/source

MATα ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1

Rothstein lab [63]

R95

MATa can1-100 his5-2 ilv3 met1 trp5-48 cdc8-1

F. Sherman lab

R803

MATa his3-513::TRP1::his3-513 leu2112::URA3::leu2-k ura3-52 trp1 cdc9

H. Klein lab [64]

R808

MATα ade1-100 his3-513::TRP1::his3-513 leu2112::URA3::leu2-k ura3-52 trp1 hpr6-1(cdc2)

H. Klein lab [64]

R809

MATα ade1-100 his3-513::TRP1::his3-513 leu2112::URA3::leu2-k ura3-52 trp1 hpr3-1 (cdcl7)

H. Klein Lab

R877

MATa ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 hdf1::LEU2

P. Jeggo lab

U739-1A

MATa ade2-1 canl-100 his3-11,15 leu2-3,112 trpl-1 ura3-1 top3-4:::URA3

Rothstein lab

W814-31D

MATa ade2-1 can1-100 his3-11,15 leu2-3,112 trpl-1 ura3-1 top1-8::LEU2

Rothstein lab

W815-8B

MATα ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 top2-4

Rothstein lab

W303-IB

a All strains obtained that originate from this study and the Rothstein lab are derivatives of W303-1B [63]. Further, can1-100, x denotes an allele of can1-100 in which an undefined secondary mutation prevents suppression by SUP4-o.

C-terminal domain (aa 450–616) required for binding the other two subunits [1]. A genetic analysis of the 70 kDa subunit was performed with the S. cerevisiae homolog, encoded by the gene RFA1 [24]. Although essential, several laboratories including our own, have isolated missense alleles of RFA1 [25-28]. These mutations display a wide range of phenotypes consistent with an in vivo role for RPA in DNA replication, repair and recombination, including UV and X ray sensitivity, growth defects, increased mutation rates and altered recombination. In particular, strains containing the rfa1-D228Y allele display increased levels of direct-repeat recombination, decreased levels of heteroallelic recombination, UV sensitivity and a S-phase delay [27]. Previous studies of this mutant have focused on its role in recombination [29]. Therefore, in this study, we have attempted to further define the role of RPA in DNA replication and repair by screening known mutants for a synthetic lethal interaction with rfa1-D228Y. Interestingly, among the replication mutants a synthetic interaction was observed with a conditional allele of the B subunit of pol α-primase, pol12-100 [30]. In addition, rfa1-D228Y strains containing a null mutation of HDF1, the S. cerevisiae homolog of the 70 kDa subunit of Ku display a delayed senescence phenotype. The analysis of telomeres in these strains reveals a synergistic reduction in telomere length in the double mutants and thus provides the first evidence of a role for RPA in telomere maintenance.

2. Materials and methods 2.1. Yeast strains and media YPD and synthetic medium were made as described previously [32, 33] with the exception that synthetic medium contains twice the amount of leucine (60 mg/L). Standard procedures were used for mating, sporulation and dissection [32]. All strains used in this study are listed in table I. 2.2. Analysis of telomere lengths To examine telomere length, genomic DNA was prepared from individual colonies on a dissection plate by the method of Hoffman and Winston [34]. The DNA samples were subsequently treated with the restriction enzyme XhoI, separated on 1% agarose gels and transferred to nitrocellulose as described by Sambrook et al. [35]. The DNA used for hybridization was poly(dG-dT)20 obtained from Boehringer Mannheim and prepared as a radiolabeled probe using a random priming kit (Pharmacia) [36]. The blots were hybridized overnight at 55 °C in 5 × Denhardts/6 × SSC and washed with 2 × SSC/0.1% SDS four times for 30 min each at 45 °C.

Characterization of genetic interactions with RFA1 3. Results 3.1. A mutation in the B-subunit of the polymerase αprimase complex is synthetically lethal with rfa1-D228Y Strains containing an rfa1-D228Y mutation display a growth defect that has been shown to result from an S phase delay [27]. The involvement of mammalian RPA in both the initiation and elongation step of DNA replication suggests that this phenotype results from a defect in DNA replication [2-4]. Biochemical studies indicate that mammalian RPA stimulates the activity of both DNA polymerase α-primase complex (pol α) and polymerase δ (pol δ), and a physical interaction has been demonstrated between the 70 kDa subunit of RPA and the primase subunits of pol α [8, 9, 11]. In the yeast S. cerevisiae, another allele of RFA1, rfa1-M2, was shown to be synthetically lethal with mutations in either the p48 primase subunit (PRI1) or catalytic subunit (CDC17) of pol α as well as the catalytic subunit of pol δ (CDC2) [25]. These results suggest that an in vivo interaction between RPA, pol α and pol δ also exists in yeast. To characterize further the replication defect of the rfa1-D228Y mutant, strains containing conditional mutations in the genes coding for the catalytic subunits of pol α (CDC17/POL1) and pol δ (CDC2/POL3) were crossed with an rfa1-D228Y strain [37-39]. Dissection of strains heterozygous for rfa1-D228Y and either cdc2 or cdc17 resulted in 4:0 segregation for viability at the permissive temperature, indicating that the rfa1-D228Y mutation is viable in both mutant backgrounds (data not shown). To confirm this result, we examined the effect of a conditional mutation in the gene that encodes the B subunit of pol α, POL12 [30]. Interestingly, lethality was observed in all cases where the rfa1-D228Y and pol12-100 alleles segregated together and at all temperatures. Furthermore, the specificity of this interaction is evidenced by the observation that mutations in several other replication factors including DNA ligase (cdc9), thymidine kinase (cdc8) and all three topoisomerases (top1∆, top2-4 and top3∆) are viable in an rfa1-D228Y background [40]. Thus, the synthetic lethality displayed by rfa1-D228Y pol12-100 double mutants validates further a genetic interaction between the p70 subunit of RPA and the pol α-primase complex and suggests that the replication defect observed in rfa1D228Y may result from an altered interaction with this polymerase. 3.2. A disruption of HDF1, the homolog of human Ku70, results in a delayed senescence phenotype in an rfa1D228Y background Ku consists of a heterodimer of polypeptides of approximately 70 and 80 kDa that avidly binds DNA ends. Cell mutants defective in Ku have been shown to be defective in double-strand break repair as well as V(D)J

73 recombination [41]. In S. cerevisiae, homologs of both the 70 and 80 kDa subunits of Ku have been identified, HDF1 and YKU80/HDF2, respectively [31, 42, 43]. A disruption of the open reading frame of either of these genes results in a temperature-sensitive phenotype for growth at 37 °C. This phenotype has been associated with a defect in DNA replication, as cells arrest with enlarged single buds and an abnormally high DNA content. These strains also display telomeres that are significantly shorter than wild-type and defects in telomere silencing [43-46]. Finally, alterations in DNA repair are evidenced by the fact that mutations in YKU70 and YKU80 enhance the radiosensitivity of rad52 [42, 43, 47]. To determine if a genetic interaction between RPA and Ku exists, an hdf1∆ strain was crossed to an rfa1-D228Y strain, sporulated and dissected. Surprisingly, at 30 °C all of the resulting hdf1∆ rfa1-D228Y segregants obtained were either non-viable or formed microcolonies that could not be propagated further. In contrast, at 23 °C, the hdf1∆ rfa1-D228Y segregants did not display a significant growth defect and were approximately the same size as the other segregants. However, when the hdf1∆ rfa1D228Y double mutants were replica-plated or streaked for single colonies, the majority of the cells were non-viable. Thus, the double mutants appear to display a progressive loss of viability or senescence phenotype, with temperature affecting the number of divisions possible. 3.3. Decreased telomere length observed in hdf1∆ rfa1D228Y double mutants A cellular senescence phenotype has been described previously for mutations in several genes involved in telomere maintenance. Strains that lack either the catalytic (EST2) or RNA subunit (TLC1) of telomerase or one of three other EST (ever shorter telomere) genes display a gradual shortening of their telomeres, and after approximately 50 to 100 divisions most of the cells in the culture stop dividing [48-50]. Since a deletion of HDF1 has been shown to be involved in telomere maintenance, it was possible that the senescence phenotype observed in hdf1∆ rfa1-D228Y double mutants was related to telomere shortening. Thus, we decided to examine telomere length in hdf1∆ rfa1-D228Y double mutants as well as in the hdf1∆ and rfa1-D228Y single mutants. Because of the limited growth capability of hdf1∆ rfa1-D228Y double mutants at 23 °C (≈25 generations), DNA was obtained directly from colonies on a dissection plate. The DNA samples were then digested with the restriction enzyme XhoI and subjected to Southern blot-hybridization analysis using the radiolabeled oligonucleotide poly(dG-dT)20 that hybridizes to telomeric repeat elements (figure 1A). XhoI cuts yeast DNA in the subtelomeric Y’ repeat that is found in many S. cerevisiae telomeres, generating a terminal restriction fragment in wild-type strains of about 1.3 kb (figure 1B), of which approximately 250 to 400 bp

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Figure 1. Telomere length in yeast strains containing an hdf1∆ and/or an rfa1-D228Y mutation. A. Structure of a typical yeast telomere. In addition to ≈250–400 bp tract of C1-3A/TG1-3 at the end of the chromosome, the terminal region of most yeast chromosomes contains a copy of the middle repetitive element X and 0–4 copies of the middle repetitive element Y’. Short stretches of C1-3A/TG1-3 are found between tandem Y’s and between X and Y’ when both exist at a single telomere. Digestion with the restriction enzyme XhoI generates a ≈1.3 kb band in wild-type strains. B. DNA was obtained directly from colonies on a dissection plate, treated with the restriction enzyme XhoI and examined by Southern analysis. Lane 1, hdf1∆; lane 2, wild-type; lane 3, hdf1∆ rfa1 D228Y; lane 4, rfa1-D228Y; lane 5, wild-type; lane 6, rfa1-D228Y; lane 7, hdf1∆ rfa1-D228Y; lane 8, hdf1∆ rfa1-D228Y.

Characterization of genetic interactions with RFA1 represents the terminal poly G1-3T tract [51]. In addition, several larger fragments are observed which correspond to terminal fragments from the subset of telomeres that lack Y′ regions (figure 1B). In rfa1-D228Y mutants, no significant defects in telomere maintenance are detected, as telomere length is similar to that observed in wild-type. In contrast, a significant reduction in telomere length is noticed in hdf1∆ strains. As previously reported, these strains display telomere lengths that are decreased by an average of approximately 100 bp (figure 1B) [43-45]. However, the shortest telomeres were observed in the hdf1∆ rfa1-D228Y double mutants, with an average reduction of 200 bp (figure 1B).The synergistic reduction observed in the hdf1∆ rfa1-D228Y double mutants suggests that Rfa1 and Hdf1 act in alternate pathways affecting telomere length and provides the first evidence of a role for RPA in telomere maintenance. Additionally, the significant reduction in telomere length observed in hdf1∆ rfa1-D228Y double mutants supports the hypothesis that the observed senescence phenotype is related to telomere shortening. 4. Discussion In our laboratory, we have isolated previously a missense allele of RFA1, rfa1-D228Y [27]. Strains containing the rfa1-D228Y allele display an increased doubling time due to a longer S-phase, which is consistent with a replication defect. In this study, we have characterized further the in vivo role of RPA in DNA replication by screening several known conditional replication mutants for a synthetic lethal phenotype in an rfa1-D228Y background. Interestingly, of all the mutants examined, only one, a conditional allele of the B subunit of pol α-primase (pol12-100) [30], displayed a genetic interaction with rfa1-D228Y. However, it should be noted that the absence of a phenotype with other replication mutants does not necessarily mean that wild-type RPA does not interact with these proteins but instead it likely reflects an allele specific interaction. The DNA polymerase α-primase complex is required for both the initiation of replication and the synthesis of Okazaki fragments on the lagging strand of the replication fork (for review see [52]). The importance of this polymerase lies in its capability of initiating DNA synthesis de novo by first synthesizing an RNA primer and then extending the primer by polymerization to produce an RNA-DNA primer. The pol α-primase complex in S. cerevisiae contains four polypeptides with apparent molecular masses of 180, 86, 58 and 48 kDa which are encoded by the essential genes POL1, POL12, PRI2 and PRI1, respectively [36, 53-55]. The largest polypeptide, p180, has been shown to be the catalytic subunit of the polymerase, while the p58 subunit has been shown to be necessary for the stability and activity of the p48 primase

75 subunit [56-59]. No enzymatic activity has been found to be associated with 86 kDa subunit. However, genetic analyses have shown that this subunit carries out an essential function at the initial stage of DNA replication [53]. Biochemical studies of the human homolog of this subunit (p70) indicate that it mediates a physical interaction between the p180 catalytic subunit and SV40 T antigen [60]. This physical interaction is functionally important since T antigen stimulation of the pol α has been shown to be dependent on the presence of the B subunit. Thus, the p86/p70 subunit appears to serve as a molecular tether, coupling the DNA polymerase α-primase complex to the DNA unwinding engine and increasing the efficiency of initiation of the first DNA chain at the origin as well as subsequent priming synthesis of DNA chains on the lagging strand template. RPA has also been shown to play an important role during the initiation and elongation steps of DNA replication [1, 52]. In initiation, RPA is involved in the formation of the primosome complex which also includes pol α and SV40 T antigen, while during elongation RPA stimulates the activity of pol α, δ and e [5-7, 9-11]. Studies carried out with the human proteins indicate that there is a physical interaction between pol α and RPA that is localized to the 70 kDa subunit of RPA and the primase subunits of the pol α complex [8]. However, the synthetic lethal interaction observed in the rfa1-D228Y pol12-100 double mutants suggests that in S. cerevisiae there may also be a physical interaction between RP-A and the B subunit. This interaction does not necessarily need to be direct, for example, as components of a common complex, the loss of both proteins may result in a destabilization that renders the complex inactive. Alternatively, since both of these proteins have been shown to be involved in the stimulation of the priming activity of pol α, the synthetic lethality may result from a cumulative defect in the ability of the pol α complex to generate primers both during the initiation and elongation steps of DNA replication. A synthetic interaction was also observed between rfa1-D228Y and a null mutation of HDF1, the S. cerevisiae homolog of the 70 kDa subunit of Ku [31]. At 23 °C, the hdf1∆ rfa1-D228Y double mutants display wild-type growth upon dissection. However, a significantly decreased level of viability is exhibited when these strains are further propagated. The observation of a similar senescence phenotype in strains defective for telomere replication combined with the previously identified role for HDF1 in telomere maintenance suggest that shortening of telomeres may be the cause of the decreased viability of the double mutant [43-46, 48-50]. This hypothesis is supported by the observation that telomere length in hdf1∆ rfa1-D228Y double mutants is reduced by 200 bp compared to rfa1-D228Y strains and 100 bp compared to hdf1∆ mutants.

76 Yeast telomeres consist of 250 to 400 base pairs of TG1-3 repeats with the G-rich strand forming the 3′ end of the chromosome [51]. During telomere replication in wild-type cells, chromosomal ends acquire transient single-stranded extensions of the G-rich strand [61]. However, it has recently been shown that, in cells lacking either HDF1 or YKU80/HDF2, single-strand extensions are now present throughout the cell cycle [62]. The fact that these extensions represent at least 30 to 50 bp of DNA suggests that one mechanism by which telomere length may be affected in hdf1∆ rfa1-D228Y mutants is increased degradation of single-stranded DNA. This hypothesis is supported by the observation that strains containing the rfa1-D228Y mutation contain decreased levels of RPA complex, the major single-stranded DNA binding protein in S. cerevisiae [27]. Thus, a reduced level of protein may result in the increased occurrence of unprotected singlestranded regions and enhanced DNA degradation. Alternatively, the decreased telomere length and synthetic lethal phenotype observed in the hdf1∆ rfa1-D228Y double mutants may result from a defect in telomere replication. It has been shown that, due to the absence of a normal terminal DNA end structure, hdf1∆ mutants are particularly sensitive to defects in components associated with telomerase. This is illustrated by the fact that cells containing a deletion of the TLC1 gene, which encodes the RNA component of telomerase, and a yku80/hdf2 mutation die after 10 generations [62]. As previously mentioned, rfa1-D228Y mutants display a defect in DNA replication as evidenced by an S-phase delay and a synthetic lethal phenotype in combination with a mutation in one of the components of the pol α complex. If telomere replication is also affected in rfa1-D228Y mutants, it is possible that in a hdf1 background this defect becomes lethal. The present study represents the first evidence for the involvement of RPA in telomere maintenance. However, we think it likely that the defect observed in rfa1-D228Y strains is important only in absence of other telomere maintenance factors. This is supported by the observation that rfa1-D228Y single mutants do not display a significant reduction in telomere length. Thus, to characterize further the role of RPA in telomere maintenance, it would be interesting to extend this analysis by investigating other RFA1 mutant alleles. Additionally, the effect of mutations in other telomere factors on the growth and telomere length of rfa1-D228Y mutants will be examined. This type of approach will allow a verification of the hypothesis that a defect in telomere maintenance is the cause of the observed senescence phenotype. Acknowledgments We thank Serge Gangloff for comments on the manuscript. This research was supported by National Institutes of Health grants GM07088 (J.S.), CA09503 (J.S.) and GM50237 (R.R.)

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