An N-terminal acidic region of Sgs1 interacts with Rpa70 and recruits Rad53 kinase to stalled forks
2012; Springer Nature; Volume: 31; Issue: 18 Linguagem: Inglês
10.1038/emboj.2012.195
ISSN1460-2075
AutoresAnna Maria Hegnauer, Nicole Hustedt, Kenji Shimada, Brietta L. Pike, Markus Vogel, Philipp Amsler, Seth M. Rubin, Fred van Leeuwen, Aude Guénolé, Haico van Attikum, Nicolas H. Thomä, Susan M. Gasser,
Tópico(s)Carcinogens and Genotoxicity Assessment
ResumoArticle20 July 2012free access An N-terminal acidic region of Sgs1 interacts with Rpa70 and recruits Rad53 kinase to stalled forks Anna Maria Hegnauer Anna Maria Hegnauer Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Faculty of Sciences, University of Basel, Basel, Switzerland Search for more papers by this author Nicole Hustedt Nicole Hustedt Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Faculty of Sciences, University of Basel, Basel, Switzerland Search for more papers by this author Kenji Shimada Kenji Shimada Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Brietta L Pike Brietta L Pike Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Markus Vogel Markus Vogel Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Philipp Amsler Philipp Amsler Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Faculty of Sciences, University of Basel, Basel, Switzerland Search for more papers by this author Seth M Rubin Seth M Rubin Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA, USA Search for more papers by this author Fred van Leeuwen Fred van Leeuwen Division of Gene Regulation, Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Aude Guénolé Aude Guénolé Department of Toxicogenetics, Leiden University Medical Center, Leiden, The Netherlands Search for more papers by this author Haico van Attikum Haico van Attikum Department of Toxicogenetics, Leiden University Medical Center, Leiden, The Netherlands Search for more papers by this author Nicolas H Thomä Nicolas H Thomä Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Susan M Gasser Corresponding Author Susan M Gasser Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Faculty of Sciences, University of Basel, Basel, Switzerland Search for more papers by this author Anna Maria Hegnauer Anna Maria Hegnauer Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Faculty of Sciences, University of Basel, Basel, Switzerland Search for more papers by this author Nicole Hustedt Nicole Hustedt Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Faculty of Sciences, University of Basel, Basel, Switzerland Search for more papers by this author Kenji Shimada Kenji Shimada Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Brietta L Pike Brietta L Pike Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Markus Vogel Markus Vogel Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Philipp Amsler Philipp Amsler Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Faculty of Sciences, University of Basel, Basel, Switzerland Search for more papers by this author Seth M Rubin Seth M Rubin Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA, USA Search for more papers by this author Fred van Leeuwen Fred van Leeuwen Division of Gene Regulation, Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Aude Guénolé Aude Guénolé Department of Toxicogenetics, Leiden University Medical Center, Leiden, The Netherlands Search for more papers by this author Haico van Attikum Haico van Attikum Department of Toxicogenetics, Leiden University Medical Center, Leiden, The Netherlands Search for more papers by this author Nicolas H Thomä Nicolas H Thomä Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Susan M Gasser Corresponding Author Susan M Gasser Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Faculty of Sciences, University of Basel, Basel, Switzerland Search for more papers by this author Author Information Anna Maria Hegnauer1,2,‡, Nicole Hustedt1,2,‡, Kenji Shimada1, Brietta L Pike1, Markus Vogel1, Philipp Amsler1,2, Seth M Rubin3, Fred van Leeuwen4, Aude Guénolé5, Haico van Attikum5, Nicolas H Thomä1 and Susan M Gasser 1,2 1Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland 2Faculty of Sciences, University of Basel, Basel, Switzerland 3Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA, USA 4Division of Gene Regulation, Netherlands Cancer Institute, Amsterdam, The Netherlands 5Department of Toxicogenetics, Leiden University Medical Center, Leiden, The Netherlands ‡These authors contributed equally to this work *Corresponding author. Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, Basel 4058, Switzerland. Tel.:+41 61 697 7255; Fax:+41 61 697 3976; E-mail: [email protected] The EMBO Journal (2012)31:3768-3783https://doi.org/10.1038/emboj.2012.195 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info DNA replication fork stalling poses a major threat to genome stability. This is counteracted in part by the intra-S phase checkpoint, which stabilizes arrested replication machinery, prevents cell-cycle progression and promotes DNA repair. The checkpoint kinase Mec1/ATR and RecQ helicase Sgs1/BLM contribute synergistically to fork maintenance on hydroxyurea (HU). Both enzymes interact with replication protein A (RPA). We identified and deleted the major interaction sites on Sgs1 for Rpa70, generating a mutant called sgs1-r1. In contrast to a helicase-dead mutant of Sgs1, sgs1-r1 did not significantly reduce recovery of DNA polymerase α at HU-arrested replication forks. However, the Sgs1 R1 domain is a target of Mec1 kinase, deletion of which compromises Rad53 activation on HU. Full activation of Rad53 is achieved through phosphorylation of the Sgs1 R1 domain by Mec1, which promotes Sgs1 binding to the FHA1 domain of Rad53 with high affinity. We propose that the recruitment of Rad53 by phosphorylated Sgs1 promotes the replication checkpoint response on HU. Loss of the R1 domain increases lethality selectively in cells lacking Mus81, Slx4, Slx5 or Slx8. Introduction The accurate replication of DNA and its segregation into daughter cells is aided by the intra-S checkpoint, which is triggered by the single-stranded DNA (ssDNA) that accumulates when DNA polymerases pause, either due to reduced nucleotide concentration or due to the presence of adducts that impair fork progression. Avoidance of fork collapse is mediated both by the Mec1/ATR kinase and by the action of a RecQ helicase, which reverses fold-back structures and resolves strand exchange to suppress inappropriate recombination events. Resumption of replication generally requires that engaged DNA polymerases remain associated with paused forks, which in wild-type yeast cells can persist for many hours (reviewed in Cobb and Bjergbaek, 2006; Tourriere and Pasero, 2007 and Aguilera and Gomez-Gonzalez, 2008). The checkpoint kinase Mec1-Ddc2 in S. cerevisiae (ATR-ATRIP in humans) plays two critical roles in this event (reviewed in Cimprich and Cortez, 2008 and Friedel et al, 2009). First, Mec1-Ddc2 regulates replisome function and enables the stable retention of replicative polymerases at very early origins like ARS607 (Cobb et al, 2003, 2005; De Piccoli et al, 2012). Second, it modifies and activates Rad53, the downstream checkpoint kinase that in turn retards cell-cycle progression, regulates levels of dNTPs and repair enzymes, represses the firing of late origins, and prevents fork collapse through poorly identified pathways (reviewed in Tourriere and Pasero, 2007; Segurado and Diffley, 2008). The activation of Mec1/Ddc2 kinase under restricted nucleotide conditions (0.2 M hydroxyurea, HU) most likely stems from the stalling of leading and/or lagging strand DNA polymerases, which generates stretches of ssDNA. These become coated by replication protein A (RPA; Aparicio et al, 1999), which signals the recruitment and activation of Mec1-Ddc2 checkpoint kinase (Zou and Elledge, 2003), not unlike the situation at resected double-strand breaks (Dubrana et al, 2007). In both budding yeast and mammals, RPA contributes to the recruitment of Mec1/ATR to stalled or damaged replication forks, through its cofactor, Ddc2/ATRIP (Melo et al, 2001; Rouse and Jackson, 2002). Intriguingly, RPA is itself a target of Mec1/ATR phosphorylation (Brush et al, 1996; Zou and Elledge, 2003). Besides RPA, fork-associated activators of the intra-S phase checkpoint include the 9-1-1 checkpoint clamp and Dbp11/TOPBP1 (Majka et al, 2006; Mordes et al, 2008; Navadgi-Patil and Burgers, 2008), while additional, unidentified co-activators are postulated to exist (Navadgi-Patil and Burgers, 2011). Once recruited Mec1/ATR phosphorylates Mrc1/Claspin, which helps activate the downstream effector kinases Rad53/CHK2, or CHK1 in mammalian cells (reviewed in Tourriere and Pasero, 2007), possibly by facilitating the contact between Mec1 and its target Rad53 (Chen and Zhou, 2009). In mammals, the RecQ helicase BLM was also reported to be a target of ATR/ATM phosphorylation, and to contribute to recovery from replicative stress (Davies et al, 2004; Rao et al, 2005). Whereas the Rad53 kinase mediates crucial downstream events in the yeast checkpoint response, Mec1/ATR has a distinct role in stabilizing replicative polymerases, particularly at early firing origins, such as the budding yeast ARS607 or ARS305. This was demonstrated by chromatin immunoprecipitation (ChIP) from cells synchronously arrested in S phase: the recovery of DNA polymerases α and ε bound to the replication fork dropped rapidly in cells lacking Mec1, but not in cells lacking Rad53 (Cobb et al, 2003, 2005). Similar separation of function was demonstrated in a study of exo1 deletion effects on viability in rad53 versus mec1 mutants (Segurado and Diffley, 2008). Nonetheless, a loss of Rad53 triggers an accumulation of both ssDNA (Sogo et al, 2002; Tourriere and Pasero, 2007) and recombination intermediates (Lucca et al, 2004). Surprisingly, and in contrast to the situation at early firing origins, it was recently reported that the replisome can be recovered largely intact and associated with later firing origins upon replication stress, in cells lacking either Mec1 or Rad53 (De Piccoli et al, 2012). These checkpoint kinases were proposed to regulate replication fork progression through multiple targets, including Psf1, a component of the replicative Cdc45-MCM-GINS helicase (De Piccoli et al, 2012). RecQ helicases have also been shown to be important for the stable binding of DNA polymerases at stalled replication forks and for efficient fork restart after exposure to HU or aphidicolin (Cobb et al, 2005; Davies et al, 2007; Bachrati and Hickson, 2008; Pirzio et al, 2008). Loss of Sgs1, the unique RecQ helicase in budding yeast, leads to a reduced recovery of DNA polymerases at early firing origins, a lower survival rate after exposure to HU (Cobb et al, 2003, 2005), and the accumulation of aberrant recombination structures after exposure to MMS (Liberi et al, 2005). Indeed, sgs1 deficient cells display abnormally high levels of recombination (Watt et al, 1996) and spontaneous gross chromosomal rearrangements (GCRs), particularly on HU (Myung and Kolodner, 2002; Schmidt and Kolodner, 2006). The role of RecQ helicases in resistance to replicative stress is conserved: mutations in three human RecQ helicases (BLM, Bloom's; WRN; Werner's, and RECQ4) cause syndromes associated with a predisposition to cancer and/or genome instability (reviewed by Bachrati and Hickson, 2008 and Ashton and Hickson, 2010). Genetic studies argue that Sgs1 acts both in complex with Top3 and Rmi1 (Gangloff et al, 1994; Chang et al, 2005; Mullen et al, 2005) and alone (reviewed in Bernstein et al, 2010). Sgs1 requires Top3 for dissolution of Holliday junctions and for enhancing DNA polymerase at stalled forks (Liberi et al, 2005; Mankouri et al, 2011), while it acts independently of Top3 to activate Rad53 in the presence of HU (Bjergbaek et al, 2005). Sgs1, like BLM and WRN, also binds Rad51 and RPA, and acts both upstream and downstream of Rad51-mediated strand invasion, to prevent and to resolve recombination intermediates. Finally, synthetic lethal screens link Sgs1 not only to recombination enzymes, but also to enzymes and proteins essential for lagging strand synthesis, such as Pol32, RNase H2 and FEN1/Rad27 (Ooi et al, 2003; Tong et al, 2004; Ii and Brill, 2005). Here, we focus on the role of Sgs1 at replication forks stalled by HU, which seems to mimic the situation that ensues when forks encounter tight DNA–protein complexes (reviewed in Aguilera and Gomez-Gonzalez, 2008). Double mutants in budding yeast have been particularly helpful in elucidating this pathway. Whereas the effects of sgs1Δ are relatively mild (Cobb et al, 2003), its combination with mec1-100, an S phase-specific mutation in Mec1, causes extensive fork collapse and a failure of nucleotide incorporation after recovery from acute exposure to HU (Cobb et al, 2005). The mec1-100 mutation compromises the intra-S phase checkpoint response, but is able to modify and activate Rad53, triggering the G2/M checkpoint (Paciotti et al, 2001). Importantly, and in contrast to the effects of mec1-100, deletion of rad53 is not additive with sgs1Δ in GCR or polymerase stability assays. Indeed, neither the loss of checkpoint activity in the rad53-11 mutant nor rad53 deletion coupled with sml1Δ affects polymerase recovery by ChIP at early firing origins (Cobb et al, 2003, 2005). The fact that Sgs1, Mec1-Ddc2 and DNA pol α all bind RPA, led us to test the hypothesis that Sgs1 influences the association of lagging strand polymerases at stalled forks through its interaction with the ssDNA binding complex. To this end, we mapped the region of Sgs1 that binds RPA and generated a mutant lacking the interaction domain, which we call sgs1-r1. We monitored the status of DNA pol α at stalled forks in mutants lacking the main RPA-interaction domain, with and without Sgs1 helicase activity. We found that the Sgs1 helicase activity and not its RPA interacting domain contributes to the stabilization of engaged DNA pol α/primase at the HU-stalled replication fork. Moreover, we show that Mec1-Ddc2 modifies Sgs1 within the RPA-interaction domain, and that once phosphorylated, Sgs1 has a significant affinity for the FHA1 phospho-threonine binding module of the downstream checkpoint kinase Rad53. We propose that the interaction of Sgs1 and Rad53 contributes to checkpoint kinase activation during replicative stress, independent of the role of Sgs1 helicase activity in stabilizing polymerases at the fork. Results Sgs1 interacts with Rpa70 through an acidic region N-terminal of the helicase domain To analyse the role of Sgs1 in stabilizing lagging strand polymerases at stalled forks, we first mapped the Sgs1–RPA interaction site. Sgs1 contains three conserved domains that are characteristic of RecQ helicases: an SF2-type helicase domain, an RQC (RecQ C-terminal) motif and an HRDC (helicase and RNase D C-terminal) domain (Figure 1A). In addition, a region of unknown structure in the N-terminus of Sgs1 (first 158 aa) has been shown to interact with Top3/Rmi1 (Bennett et al, 2000; Fricke et al, 2001; Chen and Brill, 2007; Weinstein and Rothstein, 2008). There is both an acidic block at aa 664 and a larger acidic region located N-terminal of the helicase domain. Although structure prediction suggests that this region is intrinsically disordered in solution, it has been proposed to help prevent or resolve aberrant recombination structures at MMS-treated forks (Bernstein et al, 2009). To see which domain is responsible for binding to RPA, we fused fragments containing the functional domains of Sgs1 to a B42 transactivation domain (B42-AD) and performed yeast two-hybrid (Y2H) analysis with RPA (Figure 1A). Figure 1.Mapping the interaction site between Sgs1 and Rpa70. (A) Schematic representation of Sgs1 and its functional domains. Dark and light red—largely disordered acidic region, dark red—sequences that are conserved in close homologues of S. cerevisiae; other domains labelled in figure. RQC=RecQ C-terminal motif, HRDC=helicase and RNase D C-terminal. Below are the Sgs1 domains used in Y2H experiments, which were fused to the B42 activation domain (B42-AD) in pJG46 and expressed under a galactose-inducible promoter. Numbers indicate the boundaries of the Sgs1 domains in aa. (B) Scheme of the RPA subunits with their functional domains. Rpa70 and Rpa32 were fused to the lexA-DNA binding domain (lexA-DBD) in pGAL-lexA, expressed under a galactose-inducible promoter and subjected to Y2H analysis. N-OB=N-terminal OB fold, DBD=DNA binding domain, 32 C=Rpa32 C-terminus. (C) Y2H analysis between Rpa70 fused to lexA-DBD and Sgs1 fragments fused to B42-AD was performed using a quantitative β-galactosidase assay as described in Materials and methods. Error bars indicate standard error of four or more independent transformants. (D) Y2H analysis between Rpa70 N-OB fused to lexA-DBD and Sgs1 fragments fused to B42-AD with different deletions of the three conserved regions within the RPA binding site. (E, F) Isothermal titration calorimetry (ITC) experiment of Rpa70(3–133) with Sgs1(aa 404–485) and Sgs1(aa 404–560). The dissociation constant (Kd), stoichiometry (n) and molar enthalpy (ΔH) are indicated within the figure. Download figure Download PowerPoint RPA is an evolutionarily conserved heterotrimeric protein, consisting of Rpa70, Rpa32 and Rpa14 (names based on molecular weight, or Rpa1, Rpa2 and Rpa3, based on gene names). The smallest subunit, Rpa14, is believed to mediate protein–protein interaction only within the RPA complex, while Rpa70 and Rpa32 were shown to bind other proteins (Binz et al, 2004; Zou et al, 2006). We therefore expressed full-length Rpa70 and Rpa32 fused to the LexA-DNA binding domain (LexA-DBD) under control of a galactose inducible promoter, as bait in the Y2H assay (Figure 1B). Strong interactions were scored between the largest subunit Rpa70 and the large core of the Sgs1 enzyme (aa 290–1180; Figure 1C; Supplementary Figure S1A), while Rpa32 showed a very weak β-galactosidase signal in the Y2H assay with Sgs1 (Supplementary Figure S1B). A 400-aa fragment containing only the acidic region N-terminal of the Sgs1 helicase domain bound Rpa70 as efficiently as a larger fragment (Figure 1C). Within this region, we identified three sequences of 35–41 aa by BLAST and Quick2D analysis, which are conserved among close homologues of the S. cerevisiae enzyme (dark red boxes, Figure 1; aa 404–485, aa 496–536 and aa 565–604). To test the importance of these conserved sequences for the Sgs1–Rpa70 interaction, we removed them by deleting aa 404–604 in the Sgs1-B42-AD construct. Consistently, this deletion abolished Y2H interaction between Rpa70 and Sgs1 (Figure 1C). Similarly, we mapped the interaction site on Rpa70 by fusing Rpa70 subdomains to the LexA-DBD and monitoring their interaction with the Sgs1(290–1180)-B42-AD fusion. The highest β-galactosidase activity was measured for the N-terminal oligonucleotide binding fold (N-OB) of Rpa70 without the linker region (Supplementary Figure S1A). Sgs1 bears multiple interaction sites for the Rpa70 N-OB fold To see if each conserved repeat within the Sgs1 acidic domain contributed equally to the interaction, we created three Sgs1-B42-AD deletion constructs each lacking only one of the three conserved sequences (Sgs1(290–1180, Δ404–485), Sgs1(290–1180, Δ496–536) and Sgs1(290–1180, Δ565–604)). These constructs were tested for interaction with the Rpa70 N-OB fold (Rpa70(1–133)) by Y2H analysis (Figure 1D). Deletion of the first or second conserved sequence (Sgs1(290–1180, Δ404–485) or Sgs1(290–1180, Δ496–536)) cut the β-galactosidase signal in half, while deletion of the third conserved sequence (Sgs1(290–1180, Δ565–604)) had no effect. This suggests that Sgs1 binds the RPA70 N-OB fold through two sites, aa 404–485 and aa 496–536. Indeed, deletion of the first two of the three motifs (aa 404–560 in Sgs1-B42-AD), abolished the interaction with the N-OB fold of Rpa70 almost as efficiently as deleting the entire 200 aa region, arguing that two related motifs spanning from aa 404 to 485 and aa 496 to 536, mediate the interaction with Rpa70. We confirmed that these regions of Sgs1 and Rpa70 interact directly by performing an isothermal titration calorimetry (ITC) assay with purified recombinant proteins (Figure 1E and F). We found that both Sgs1(404–485) and Sgs1(404–560) bound RPA70(3–133) with similar affinity (Kd=70±20 μM and Kd=34±1 μM, respectively). The ITC data suggested differences in the complex stoichiometry (n) and molar enthalpy (ΔH) between the two Sgs1 fragments: it appears that the more N-terminal motif of Sgs1 (aa 404–485) binds one molecule of the RPA70 N-OB fold, while the larger domain (aa 404–560) is able to bind two. This suggests that Sgs1 might be able to bind multiple RPA complexes, possibly leading to RPA delivery and/or removal as Sgs1 unwinds duplex DNA. The Sgs1–RPA interaction site is structurally isolated from the helicase domain and its deletion does not affect protein stability To determine whether this region of Sgs1 is also important for interaction with RPA in vivo, we deleted amino acids 404–604 within the SGS1 chromosomal locus using a PCR-based allele-replacement technique. The resulting allele (sgs1-r1; Figure 2A), C-terminally tagged by 13Myc, is expressed when tested by western blot analysis on whole cell extracts (Figure 2B and C, inputs). The signal for sgs1-r1–13Myc is slightly stronger than for wild-type Sgs1–13Myc, which may either reflect a better blotting efficiency or slightly improved stability. This deletion leaves intact the aa 664 shown to be important for resolving recombination intermediates, and is distinct from the previously published AR2 deletion, which reduced Sgs1 levels (Bernstein et al, 2009). Figure 2.Loss of the acidic region in Sgs1 impairs Sgs1–RPA interaction in vivo. (A) Schematic representation of sgs1-r1: a new allele generated by deleting the acidic region (aa 404–604) at the endogenous SGS1 locus. The deleted region is indicated by a dashed line. Black=acidic block, dark blue=helicase domain, light blue=RQC domain, blue=HRDC domain. (B) Wild type and sgs1-r1 were C-terminally fused to 13Myc epitopes at the endogenous SGS1 locus (Sgs1–13Myc; GA-5311, sgs1-r1–13Myc; GA-5313) and expression levels were analysed by western blot with anti-Myc antibody. Non-tagged strain (GA-7249) was used as a negative control. Anti-actin was used to detect Act1 as a loading control. (C) Co-IP of exponentially growing 13Myc-tagged Sgs1 (GA-1759) or sgs1-r1 (GA-5316) with 3HA-tagged Rpa70. Exponentially growing cells were collected for IP using Dynabeads either coupled to monoclonal anti-Myc (AB) or not (ctrl). Western blots were probed with anti-Myc (9E10) for Sgs1 or sgs1-r1 and anti-HA (F-7) for Rpa70. (D) sgs1-r1 (GA-4848) and srs2Δ (GA-1805), as well as sgs1-hd (GA-5445) and srs2Δ (GA-5334) mutants were crossed and sporulated and tetrad analysis was performed. (E) Ten-fold serial dilutions of the following strains were plated onto YPAD, ±20 mM or 100 mM HU, 0.005% or 0.033% MMS: GA-1981 (WT), GA-4978 (mec1-100), GA-5457 (sgs1Δ), GA-4967 (sgs1Δ mec1-100), GA-5076 (sgs1-r1), GA-5077 (sgs1-r1 mec1-100), GA-5445 (sgs1-hd) and GA-5447 (sgs1-hd mec1-100). Download figure Download PowerPoint To monitor the interaction of the sgs1-r1 mutant protein with RPA in vivo, we performed co-immunoprecipitation (co-IP) experiments with appropriately tagged proteins. Strains expressing Rpa70–3HA and either 13Myc-tagged Sgs1 or sgs1-r1 were released from G1 phase for 20 min to allow cells to accumulate in S phase. Rpa70–3HA and Sgs1-13Myc were efficiently precipitated as a complex using either anti-Myc antibody (Figure 2C) or anti-HA (Supplementary Figure S2). In the sgs1-r1–13Myc precipitation, the efficiency of Rpa70 recovery was reduced roughly two-fold (Figure 2C), as was the reciprocal recovery (sgs1-r1 by Rpa70, Supplementary Figure S2). This suggests that the deleted domain indeed mediates Sgs1–RPA interaction in vivo, although other contacts may support interactions in the context of the holo-RPA complex. Indeed, residual binding could be explained by the interaction detected between Sgs1 and Rpa32 (Supplementary Figure S1B), through a site unaffected by the sgs1-r1 mutation, or by an indirect interaction of sgs1-r1–13Myc and Rpa70–3HA to DNA. To ensure that the sgs1-r1 protein retained helicase activity, despite the reduced interaction with RPA, we tested the ability of sgs1-r1 to support growth in a srs2 null background. Rothstein and colleagues have shown that the helicase activity of Sgs1 is essential for growth in the absence of the Srs2 helicase (Weinstein and Rothstein, 2008). Tetrad analysis confirms that spores containing helicase-dead Sgs1 (K706R or sgs1-hd) in combination with srs2Δ showed almost no growth, while the sgs1-r1 srs2Δ double mutant spores grew normally (Figure 2D). We conclude that sgs1-r1 retains helicase activity, consistent with its weak suppression of top3Δ slow growth (Supplementary Figure S3). We next tested whether the sgs1-r1 allele yields the same levels of sensitivity to DNA damage as sgs1Δ in drop assays. This assay monitors the capacity of cells to repair DNA damage induced by different genotoxic drugs. Previous work has shown that sgs1Δ is sensitive to low concentrations of the replication fork inhibitor HU and the alkylating agent MMS, and that this phenotype is dramatically enhanced when sgs1Δ is coupled to the S phase-specific Mec1 mutant, mec1-100 (Cobb et al, 2005). As described previously, we observed that both sgs1Δ and the helicase-dead mutant sgs1-hd are sensitive to HU and MMS, and show synthetic sensitivity when combined with the mec1-100 allele (Figure 2E). This was not observed for the sgs1-r1 mutant, although it did show a mild sensitivity to high concentrations of MMS (0.033% MMS, Figure 2E). Surprisingly, no additive effects were seen when sgs1-r1 mec1-100 cells were compared with the single mec1-100 mutant, suggesting that either sgs1-r1 acts on the same pathway as mec1-100 or it simply does not affect survival during persistent replicative stress (Figure 2E). The Sgs1 helicase function, but not its R1 domain, stabilizes DNA pol α on HU Given that fork restart after prolonged exposure to HU requires different activities than does growth under persistent damage, we next arrested S-phase cells in 0.2 M HU for 2–6 h, and quantitatively measured cell survival after plating on HU-free YPAD (Figure 3A). Similar to the lack of sensitivity observed on HU-containing plates, we found that sgs1-r1 did not confer sensitivity to this acute HU treatment. Unlike the sgs1Δ mec1-100 or sgs1-hd mec1-100 double mutants (Figure 3A), the sgs1-r1 mec1-100 combination showed no additive or synergistic effects. This suggested that strong RPA-Sgs1 binding is not crucial for fork recovery after HU-induced replication fork arrest. Figure 3.sgs1-r1 does not destabilize DNA pol α from HU-stalled replication forks in contrast to a helicase-deficient sgs1 mutant. (A) Recovery from replication fork stalling was monitored as colony outgrowth of cells which were synchronized in G1 by α-factor arrest and released into S phase in the presence of 0.2 M HU for the indicated times. Strains used were GA-1981 (WT), GA-180 (WT rad5-535), GA-5457 (sgs1Δ), GA-4978 (mec1-100), GA-4967 (sgs1Δ mec1-100), GA-5076 (sgs1-r1), GA-4504 (sgs1-r1 mec1-100 rad5-535), GA-5445 (sgs1-hd) and GA-5447 (sgs1-hd mec1-100). 0 min after release indicates G1 phase. (B, C) ChIP was performed on synchronized cultures released into S phase in the presence of 0.2 M HU. 3HA-tagged DNA pol α was precipitated with monoclonal anti-HA antibody (F-7) coupled to Dynabeads. Strains used were GA-4973 (WT), GA-4974 (mec1-100), GA-5055 (sgs1-r1), GA-5075 (sgs1-r1 mec1-100), GA-5449 (sgs1-hd) and GA-5451 (sgs1-hd mec1-100). The ChIP data for sgs1Δ and sgs1Δ mec1-100 from Cobb et al (2005) are shown for comparison (indicated by the dashed lines). The relative enrichment for ARS607 or ARS522 was obtained by normalizing the absolute enrichment at ARS607 or ARS522 for each time point to the absolute enrichment at a locus 14 kb away from ARS607. Mutant strains are indicated using the same colour code as in (A). (D) ChIP was performed as described above using strains GA-4973 (WT), GA-6266 (sgs1-r1-hd), GA-6260 (sgs1-r1-hd mec1-100) and GA-4975 (sgs1Δ mec1-100). (E) Primers (grey bars) used for ChIP that amplify the genomic regions corresponding to the early firing origin ARS607, a region 14 kb away from ARS607 (+14 kb) and the late firing origin ARS522 are shown. Download figure Download PowerPoint We next tested the effects of sgs1-r1 on DNA pol α association at replication forks arrested on HU. We performed ChIP for DNA pol α after synchronizing single and double mutants in G1 and releasing them into S phase in the presence of 0.2 M HU. Over 1 h, the abundance of DNA-bound DNA pol α was quantified by real-time PCR analysis of the recovered DNA, using prime
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