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Role of Saw1 in Rad1/Rad10 complex assembly at recombination intermediates in budding yeast

2013; Springer Nature; Volume: 32; Issue: 3 Linguagem: Inglês

10.1038/emboj.2012.345

1460-2075

Fuyang Li, Junachao Dong, Robin Eichmiller, Cory Holland, Eugen C. Minca, Rohit Prakash, Patrick Sung, Eun Yong Shim, Jennifer A. Surtees, Sang Eun Lee,

Carcinogens and Genotoxicity Assessment

Article8 January 2013free access Source Data Role of Saw1 in Rad1/Rad10 complex assembly at recombination intermediates in budding yeast Fuyang Li Fuyang Li Department of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USACo-first author. Search for more papers by this author Junachao Dong Junachao Dong Department of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USACo-first author. Search for more papers by this author Robin Eichmiller Robin Eichmiller Department of Biochemistry, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, NY, USACo-first author. Search for more papers by this author Cory Holland Cory Holland Department of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA Search for more papers by this author Eugen Minca Eugen Minca Department of Biochemistry, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, NY, USA Search for more papers by this author Rohit Prakash Rohit Prakash Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT Search for more papers by this author Patrick Sung Patrick Sung Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT Search for more papers by this author Eun Yong Shim Eun Yong Shim Department of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA Division of Radiation Biology, Department of Radiation Oncology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA Search for more papers by this author Jennifer A Surtees Corresponding Author Jennifer A Surtees Department of Biochemistry, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, NY, USA Search for more papers by this author Sang Eun Lee Corresponding Author Sang Eun Lee Department of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA Division of Radiation Biology, Department of Radiation Oncology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA Search for more papers by this author Fuyang Li Fuyang Li Department of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USACo-first author. Search for more papers by this author Junachao Dong Junachao Dong Department of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USACo-first author. Search for more papers by this author Robin Eichmiller Robin Eichmiller Department of Biochemistry, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, NY, USACo-first author. Search for more papers by this author Cory Holland Cory Holland Department of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA Search for more papers by this author Eugen Minca Eugen Minca Department of Biochemistry, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, NY, USA Search for more papers by this author Rohit Prakash Rohit Prakash Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT Search for more papers by this author Patrick Sung Patrick Sung Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT Search for more papers by this author Eun Yong Shim Eun Yong Shim Department of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA Division of Radiation Biology, Department of Radiation Oncology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA Search for more papers by this author Jennifer A Surtees Corresponding Author Jennifer A Surtees Department of Biochemistry, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, NY, USA Search for more papers by this author Sang Eun Lee Corresponding Author Sang Eun Lee Department of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA Division of Radiation Biology, Department of Radiation Oncology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA Search for more papers by this author Author Information Fuyang Li1, Junachao Dong1, Robin Eichmiller2, Cory Holland1, Eugen Minca2, Rohit Prakash3, Patrick Sung3, Eun Yong Shim1,4, Jennifer A Surtees 2 and Sang Eun Lee 1,4 1Department of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA 2Department of Biochemistry, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, NY, USA 3Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 4Division of Radiation Biology, Department of Radiation Oncology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA *Department of Biochemistry, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, 3435 Main Street, Buffalo, NY 14214, USA. Tel:+1 716 829 6083; Fax:+1 716 829 2725; E-mail: [email protected] Se Lee, Department of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA. Tel:+1 210 562 4157; Fax:+1 210 562 4161; E-mail: [email protected] The EMBO Journal (2013)32:461-472https://doi.org/10.1038/emboj.2012.345 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 The Saccharomyces cerevisiae Rad1/Rad10 complex is a multifunctional, structure-specific endonuclease that processes UV-induced DNA lesions, recombination intermediates, and inter-strand DNA crosslinks. However, we do not know how Rad1/Rad10 recognizes these structurally distinct target molecules or how it is incorporated into the protein complexes capable of incising divergent substrates. Here, we have determined the order and hierarchy of assembly of the Rad1/Rad10 complex, Saw1, Slx4, and Msh2/Msh3 complex at a 3′ tailed recombination intermediate. We found that Saw1 is a structure-specific DNA binding protein with high affinity for splayed arm and 3′-flap DNAs. By physical interaction, Saw1 facilitates targeting of Rad1 at 3′ tailed substrates in vivo and in vitro, and enhances 3′ tail cleavage by Rad1/Rad10 in a purified system in vitro. Our results allow us to formulate a model of Rad1/Rad10/Saw1 nuclease complex assembly and 3′ tail removal in recombination. Introduction Environmental and metabolic stress induces DNA damage that interferes with essential DNA transactions such as DNA replication and chromosome segregation. Multiple mechanisms and their affiliated DNA metabolizing enzymes are responsible for sensing and repairing DNA damage efficiently and faithfully. The yeast Rad1/Rad10 complex is a nuclease that participates in multiple repair pathways including nucleotide excision repair (NER), base excision repair (BER), inter-strand crosslink repair (ICLR), and homologous recombination (HR) (reviewed in Ciccia et al, 2008). In NER, Ssl2/Rad3 (XPB/D in humans) unwinds DNA containing DNA lesions, Rad1/Rad10 (XPF/ERCC1 in humans) then incises the damaged DNA strand on the 5′ side, concurrently with a 3′ incision made by Rad2 (XPG in humans), producing a gapped DNA intermediate that can be filled by a DNA polymerase (Sijbers et al, 1996; de Boer and Hoeijmakers, 2000; Prakash and Prakash, 2000). In HR, Rad1/Rad10 (and XPF/ERCC1) cleaves 3′ non-homologous tails after annealing of flanking repeats in single-strand annealing (SSA) or from the D-loop structure that harbours an invading DNA end with a non-homologous sequence (Schiestl and Prakash, 1990; Fishman-Lobell and Haber, 1992; Tomkinson et al, 1993; Adair et al, 2000). Removal of the 3′ non-homologous DNA tail is a prerequisite for the initiation of repair DNA synthesis. In BER, Rad1/Rad10 plays a redundant or back-up role to Apn1, Apn2, and Mus81/Mms4, since any of the above can cleave a 3′ blocked end as a 3′-flap endonuclease (Boiteux and Guillet, 2004; Guzder et al, 2004). In ICLR, Rad1/Rad10 is hypothesized to either unhook the crosslink or to remove a non-homologous DNA tail to restore the replication fork by HR (Bergstralh and Sekelsky, 2008). The versatility of Rad1/Rad10 for a wide range of DNA lesions and structures poses a unique question: how does Rad1/Rad10 recognize these different substrate molecules and how is its nuclease activity regulated to enhance the specificity for these molecules? The available evidence has hinted that substrate specificity of Rad1/Rad10 might be dictated by its interaction partners, some of which recruit and position Rad1/Rad10 at the cleavage target. For instance, Rad14 (XPA in mammals) interacts with Rad1/Rad10 and targets it to UV lesions (Li et al, 1995; Guzder et al, 2006). However, the mechanisms for targeting Rad1/Rad10 (and XPF/ERCC1) to recombination, BER substrates and intermediates have not been determined. Rad1/Rad10 interacts with many proteins during HR, some of which may target Rad1/Rad10 to recombination intermediates, while others may modulate the enzyme activity or the substrate stability. The Rad52 and Rad59 proteins likely contribute to 3′ tail cleavage by mediating the annealing of complementary single-stranded DNA (ssDNA) strands, a prerequisite for Rad1/Rad10 targeting (Lyndaker and Alani, 2009). In human cells, Rad52 directly interacts with ERCC1, and this interaction stimulates XPF/ERCC1 activity in processing 3′ non-homologous tailed substrates (Motycka et al, 2004). Msh2/Msh3 was initially proposed to recognize recombination intermediates containing a 3′-flap, thereby recruiting Rad1/Rad10 to the 3′ tail or stabilizing the complex of Rad1/Rad10 and DNA (Kirkpatrick and Petes, 1997). However, inactivating Msh2 or Msh3 has no effect on SSA between longer repeats (>1 kb). Instead, a role for Msh2/Msh3 in stabilizing annealed intermediates prior to 3′ tail cleavage has been invoked (Sugawara et al, 1997). Slx4 is a subunit of the Slx1/Slx4 complex that was originally identified in a genetic screen for synthetic lethality with the Sgs1 helicase. In addition, it has recently been implicated in SSA and 3′ non-homologous tail removal independent of Slx1 (Mullen et al, 2001; Flott et al, 2007). Interestingly, Slx4 is a phosphoprotein and its phosphorylation depends on DSBs and on the Mec1 and Tel1 kinases (Flott and Rouse, 2005; Flott et al, 2007). DNA damage-induced phosphorylation of Slx4 plays an essential role in SSA repair of DNA breaks but the precise mechanism is not yet known (Toh et al, 2010). Recently, Slx4 has been implicated in targeting ERCC1-XPF to ICL repair substrates by physical interaction (Andersen et al, 2009; Fekairi et al, 2009; Munoz et al, 2009; Svendsen et al, 2009; Crossan et al, 2011; Kim et al, 2011; Stoepker et al, 2011). Finally, Saw1 was identified from a genetic screen for novel proteins involved in SSA and was shown to physically interact with multiple factors involved in 3′ tail cleavage (Li et al, 2008). Saw1 is a strong candidate for targeting Rad1/Rad10 to 3′ tailed recombination intermediates because deletion of SAW1 or expression of saw1 mutants deficient in interaction with Rad1/Rad10 abrogates binding of Rad1/Rad10 at 3′ tails and blocks SSA (Li et al, 2008). The recent identification of several new factors required for 3′ tail cleavage during HR raises questions about the precise function of these factors in Rad1/Rad10-mediated 3′ tail removal and the coordination among these proteins. How is the recruitment and assembly on the DNA substrate regulated and properly orchestrated to produce the specific cleavage pattern? Indeed, what are the components of the protein machinery that mediates 3′ tail removal? We have approached the above questions by determining the temporal and spatial protein recruitment patterns at 3′-flap recombination intermediates and also their association hierarchy using chromatin immunoprecipitation (ChIP) and live-cell imaging. We have assessed the DNA binding activity of Saw1 using electrophoretic mobility shift assay (EMSA) and by ChIP. We demonstrate that Saw1 contributes to 3′ tail removal as a mediator between a 3′ DNA flap substrate and Rad1/Rad10. Collectively, our results suggest a model for how the 3′ tail removal machinery is assembled onto DNA and shed light on the molecular steps in processing 3′ tailed DNA intermediates. Results Recruitment of nuclease deficient rad1 to 3′ tailed recombination intermediates requires Saw1, Msh2, but does not Slx4 In vivo studies have shown that 3′ tail removal during recombination requires at least 12 proteins: Rad1, Rad10, Msh2, Msh3, Rad52, Rad59, Saw1, Slx4, Srs2, and Rpa1, 2, and 3. All but Saw1 have a known DNA binding activity and likely target, position, and/or regulate Rad1/Rad10-mediated 3′-flap cleavage. To elucidate their role(s) in the early steps of 3′ tail removal, we examined Rad1 recruitment to a 3′ tail in strains lacking Rad52, Rad10, Msh2, Saw1, or Slx4. Association of nuclease proficient Rad1 at 3′ tails in wild-type cells was undetectable by ChIP, presumably due to the rapid dissociation of Rad1/Rad10 following cleavage (see Supplementary Figure S3A). We therefore used ChIP to examine the association of nuclease-deficient variants of Rad1 (rad1nuc-) at the 3′-flap after the HO-endonuclease-mediated induction of a DSB that is flanked by 205 bp of ura3 repeats (Li et al, 2008) (Figure 1B). We predicted that the nuclease-deficient rad1 would stably associate with the target DNA to allow for its detection by ChIP. Figure 1.Recruitment of rad1nuc- to 3′ non-homologous tail carrying recombination intermediates. (A) Domain structure of Rad1 and the location of rad1 mutations that disrupt nuclease activity. (B) Schematic illustration of HO break and the flanking repeats in tNS1379/EAY1141 or YMV80 strain used for ChIP assay. Location of primers and the size of the repeats are shown. Paired arrows indicate primers used for ChIP assay. (C, D) The requirements of Rad10, Rad52, Msh2, Saw1, and Slx4 for the recruitment of rad1nuc- to the proximal (pJC1 and pJC2) (C) or distal (pJC3 and pJC4) (D) side of the 3′-flap site in tNS1379 strain. Association of rad1nuc- to the 3′-flap 6 h post HO induction in the msh2Δ derivative of YMV80 strains is also shown (D). Fold enrichment represents the ratio of the rad1 IP PCR signal before and after HO induction, normalized by the PCR signal of the MAT control. Data represent the mean±s.d. of three or more independent experiments. Download figure Download PowerPoint To this end, we generated three rad1 mutants, all of which carry amino-acid substitutions at putative nuclease active sites that are conserved from yeast to human (Enzlin and Scharer, 2002) (Figure 1A). Two of the three mutant proteins (D825A, K865A) were purified and biochemically confirmed to be deficient in 3′-flap cleavage despite retaining DNA-binding activity (Supplementary Figure S1D). Expression of the mutant rad1 proteins in rad1Δ did not alleviate UV sensitivity (due to impaired NER) or restore SSA repair of a DNA break flanked by 205-bp ura3 repeats (Supplementary Figure S2). We also confirmed that these rad1 variants were expressed at the normal level and retain physical interactions with both Rad10 and Saw1 (Supplementary Figure S3B). Most importantly, all three rad1 mutants exhibited strong enrichment at 3′ tails within 1 h after HO expression and remain associated for up to 4 h, providing direct evidence that nuclease deficiency results in Rad1 accumulation at 3′ tailed intermediates (Supplementary Figure S3C and D). We then used ChIP to examine the effect of deleting individual 3′ tail removal genes on the retention of rad1 mutants (rad1nuc-) at the HO break. We discovered that recruitment of rad1nuc- relies on Rad10, Rad52, and Saw1 (Figure 1C and D). The results suggest that dimerization with Rad10, annealing of complementary ssDNA by Rad52, and targeting of Rad1/Rad10 nuclease to recombination intermediates by Saw1 are important for 3′ non-homologous tail removal complex assembly. Interestingly, the effect of MSH2 gene deletion on rad1nuc- recruitment is complex and depends on the length of repeats; when repeats are 205-bp long, Msh2 is required (Figure 1C and D), but when they are 1-kb long, Msh2 is not needed (see Figure 1D, YMV80). The results are consistent with Msh2 not being required for SSA between 1-kb repeats (Supplementary Figure S4) and with the proposed role for Msh2/Msh3 in stabilizing products made by annealing short complementary DNA strands (Sugawara et al, 1997). Slx4 is implicated in Rad1-dependent 3′ non-homologous tail cleavage and SSA independently of Slx1 (Flott et al, 2007; Li et al, 2008; Lyndaker et al, 2008). We discovered that rad1nuc- is recruited to recombination intermediates in slx4Δ cells with kinetics similar to wild type (Figure 1C and D). Interestingly, the recruitment of Rad1 is slightly faster on the proximal side of the break in slx4 mutant (Figure 1C). The results suggest that Slx4 is dispensable for Rad1 recruitment. Collectively, the results suggest that recruitment of Rad1 to 3′ tails requires the combined action of multiple tail removal factors, but not Slx4. Recruitment of Msh2 to 3′ non-homologous tails depends on Rad52, but not other tail removal factors We have previously shown that Msh2/Msh3 binds branched substrates in vitro, and hypothesized that it enhances the accessibility of DNA to cleavage by Rad1/Rad10 (Surtees and Alani, 2006). To test this model, we used ChIP to monitor Msh2 recruitment at 3′ tails formed by 205-bp ura3 or 1-kb leu2 repeats flanking the HO break. Msh2 (tagged with four HA epitopes) was rapidly recruited to both sides of the recombination intermediate, as previously observed (Evans et al, 2000; Lyndaker and Alani, 2009), whereas the deletion of RAD52 abrogated this binding (Figure 2). The deletion of RAD1, RAD10, SAW1, or SLX4 actually enhanced binding of Msh2 to the DNA tails, although the kinetics of localization appear altered in slx4Δ. We reasoned that enhanced Msh2 binding in these mutants stems from the lack of tail removal causing persistent accumulation of Msh2 (Evans et al, 2000; Lyndaker and Alani, 2009). Based on the above results, we could deduce that the assembly of tail removal complex requires binding of Rad52, then Msh2/Msh3, and is finally completed with the Saw1-dependent recruitment of Rad1/Rad10. Figure 2.Recruitment of Msh2 (A, B) and Saw1 (C, D) to 3′ non-homologous tail carrying recombination intermediates. The recruitment of Msh2 to the centromere distal (A) or the proximal (B) side of the 3′-flap in EAY1141 or its mutant derivatives. The recruitment of Saw1 to the proximal (C) or distal (D) side of the 3′-flap in the EAY1141 strain expressing wild-type or mutant saw1 proteins using ChIP assays. Fold enrichment is calculated as described in Figure 1. Data represent the mean±s.d. of three or more independent experiments. Download figure Download PowerPoint Saw1 associates with 3′-flaps in vivo The requirement of Saw1 for rad1nuc- recruitment to 3′ tails could be mediated through interaction between Saw1 and the DNA substrate. To address this question, we used ChIP to ask whether Saw1 associates with 3′ tailed intermediates. We found that Saw1 is recruited to 3′ tails with kinetics similar to that of rad1nuc- (Figure 2C and D). We also examined Saw1 association at 3′-flaps in strains deleted for one of the other 3′ tail removal genes. We found that recruitment of Saw1 also depends on RAD52 and MSH2 (Figure 2C and D). Again, Slx4 is dispensable for enrichment of Saw1 at the 3′ tailed DNA. Finally, association of Saw1 with the 3′ intermediate is abolished by a mutation (saw1DB-) that disrupts its DNA-binding activity (Figure 2C and D, and see below). Importantly, this mutation prevents recruitment of rad1nuc- to the 3′ tails (Figure 5B). These results thus revealed that Saw1 targets Rad1/Rad10 to 3′ tailed recombination intermediates. Saw1 is a structure-specific DNA binding protein The Saw1 ChIP experiments indicated that an interaction between Saw1 and the DNA intermediate is critical for recruiting Rad1/Rad10 and for the completion of SSA. To define Saw1's DNA-binding activity biochemically, we expressed Saw1 bearing a 6 × His tag in E. coli and purified it to near homogeneity (Figure 3A). Purified Saw1 interacts with Rad1 and Rad52, indicating that it is functional (Supplementary Figure S5A). We tested purified Saw1 for DNA binding in a DNA mobility shift assay with a variety of radiolabelled substrates. The results showed that Saw1 has the highest affinity for 5′- or 3′-flap DNA, a splayed arm structure, and a replication fork-like structure, but binds single-stranded DNA and 5′ or 3′ tailed DNA only weakly, and has no affinity for linear duplex DNA (Figure 3B). The presence of Saw1 in the nucleoprotein complex was validated by an antibody super-shift experiment using anti-6 × His or anti-Saw1 antibody and the splayed Y structure as substrate; antibody alone did not affect the DNA mobility (Supplementary Figure S5B). Saw1 also binds to a DNA substrate containing 14 bp bubble structure as efficiently as the splayed Y structure. Saw1 however did not bind to a substrate with 7 bp bubble or a hairpin with either a 10 base or 20 base loop (Supplementary Figure S6). The results thus revealed a structure-specific DNA binding activity in Saw1. Figure 3.Saw1 binds to DNA with the distinct structural motif. (A) 6 × His-tagged Saw1 protein was purified from E. coli as described in Materials and methods. Purified proteins were analysed by SDS–PAGE followed by Coomassie blue staining. (B) Electrophoretic mobility shift assay (EMSA) for 6 × His-Saw1. Reactions contained the indicated 32P-labelled substrates (0.1 pmol) and purified 6 × His-Saw1 (200 nM). Reactions were incubated on ice for 30 min, and protein–DNA complexes were analysed by 6% PAGE followed by autoradiography. 32P labels are indicated with asterisks. The splayed arm, 3′ and 5′ tail, 3′- and 5′-flap DNA all contain single-stranded DNA flap of 30 nucleotides. (C) EMSA to determine Saw1 binding to DNA substrates with various length of 3′-flaps. The indicated DNA substrates were incubated with or without purified Saw1 protein (10–200 nM) and the DNA-Saw1 complex was detected by acrylamide mini gel electrophoresis followed by autoradiography. Data were assembled by removal of irrelevant lanes but from the same gel or from multiple gels exposed in parallel.Source data for this figure is available on the online supplementary information page. Source Data for Figure 3A [embj2012345-sup-0001-SourceData-S1.jpg] Source Data for Figure 3B [embj2012345-sup-0002-SourceData-S2.jpg] Source Data for Figure 3C [embj2012345-sup-0003-SourceData-S3.jpg] Download figure Download PowerPoint Previous genetic studies demonstrated that a 3′ DNA tail is efficiently cleaved by Rad1/Rad10 only if it is longer than 20 nucleotides (Colaiacovo et al, 1999; Li et al, 2008). We therefore investigated whether Saw1's affinity to 3′-flap DNA as a function of the length of the 3′ tails (Figure 3C). We found that the Saw1 binds to a 3′-flap only when the flap strand is at least 10 nucleotides in length, and that the protein clearly has a higher affinity for substrates with a longer flap strand. DNA binding of Saw1 is required for Rad1/Rad10-dependent 3′-flap removal To explore the biological significance of the structure-specific DNA binding activity of Saw1 in Rad1/Rad10-dependent 3′-flap cleavage, we screened for saw1 mutants that are deficient in DNA binding but retain physical interactions with Rad1, Msh2, and Rad52. To help identify such mutants, we aligned Saw1 and its fungal homologues. This analysis revealed high conservation of six positively charged amino acids at the carboxyl terminus of Saw1 across species (Supplementary Figure S7). We reasoned that these basic residues could be involved in DNA binding via interactions with the phosphodiester backbone of DNA. To test this premise, we produced saw1 mutants deleting these conserved amino acids or the surrounding amino acids (Materials and methods). The resulting saw1 mutants bearing 6 × His tag (saw1-Δ192–195, -Δ202–220, -Δ221–229, and -Δ244–250; Supplementary Figure S8B) were tested for their DNA binding by EMSA using 3′-flap DNA as substrate (Figure 4; Supplementary Figure S8C). Included in the EMSA were the saw1 mutants with deletions or substitution of the conserved amino acids at the N-terminus (saw1Δ18–24, or saw1-R19A, respectively) that were shown to have an impaired interaction with Rad1 (Li et al, 2008; Supplementary Figure S9A). Among the saw1 mutants, the variant lacking the six positively charged C-terminal residues (saw1-Δ244–250 or saw1DB-) is the only one that is almost completely devoid of 3′-flap DNA binding. GST-saw1DB- retains the ability to interact with TAP-tagged Rad1, Msh2, and Rad52 as determined in a GST pull-down experiment (Supplementary Figure S8A). We next tested the effect of the Δ244–250 mutation on 3′-flap cleavage in cells (Sugawara et al, 2000; Li et al, 2008). Importantly, we used two distinct assays testing either the HR mediated plasmid retention (Supplementary Figure S9B) or chromosome-based SSA (Supplementary Figure S9C), respectively, both of which depend on 3′ non-homologous tail removal. In both, the saw1DB- mutation causes a severe defect in Rad1/Rad10-dependent 3′-flap cleavage. The results thus provide genetic evidence that the structure-specific DNA binding activity of Saw1 is indispensable for Rad1/Rad10-mediated 3′-flap processing. Figure 4.Characterization of saw1 mutant defective in interaction with Rad1 or DNA binding. The 3′-flap DNA binding, physical interaction with Rad1, and efficiency of 3′-flap removal were assessed by EMSA, GST pull-down, and plasmid-based 3′-flap removal assays, respectively, as described in Materials and methods. Percent 3′-flap removal efficiency was calculated by scoring % plasmid retention in strains deleted for SAW1 supplemented with wild-type Saw1, or mutant saw1 proteins. NA, not available. Download figure Download PowerPoint Saw1 is required for recruitment of Rad1/Rad10 to 3′-flaps In order to determine whether Saw1 facilitates the binding of Rad1/Rad10 to 3′-flaps, we co-expressed 6 × His-Rad1, Rad10, and 6 × His-Saw1 or 6 × His-saw1 mutants in E. coli. We co-purified 6 × His-Rad1/Rad10 with 6 × His-Saw1 or saw1 mutant derivatives through a Cobalt agarose column. The three proteins co-eluted (Supplementary Figure S10) and the eluate was used to perform DNA gel mobility shift experiments. The strategy allowed us to purify His-Saw1 or His-saw1 mutants independent of its interaction with Rad1/Rad10 complex. Purified Rad1/Rad10 did not bind the 3′-flap DNA but became incorporated into a higher order nucleoprotein complex when Saw1 was present (Figure 5A). In contrast, fewer higher order species was seen when saw1-Δ18–24 or saw1DB- was used (Figure 5A; Supplementary Figure S11). The results provide support for the model that Saw1 targets Rad1/Rad10 to the 3′ tailed substrate in HR. Figure 5.Saw1 recruits Rad1/Rad10 complex to 3′-flap DNA substrate. (A) His-Rad1-Rad10 and His-Saw1 were co-expressed from E. coli and purified together with wild-type His-Saw1, His-saw1-Δ18–24, or His-saw1DB-, respectively, over a Cobalt column. The 5′-32P-labelled 3′-flap DNA substrate (0.1 pmol) was then incubated with increasing amounts of the His-Rad1/Rad10/His-Saw1 mixture, reported as μg/ml because the stoichiometry of any complexes is unclear The reaction mixtures were separated by acrylamide mini gel electrophoresis followed by autoradiography to detect Rad1/Rad10/Saw1-DNA or Saw1-DNA complex. Data were assembled by removal of irrelevant lanes and from two gels exposed in parallel. (B) The recruitment of rad1nuc- to the proximal to the 3′-flap site in EAY1141 and its saw1-R19A or saw1DB- mutant derivatives was examined by ChIP assays. Fold enrichment is calculated as described in Figure 1. Data represent the mean±s.d. of three or more independent experiments. (C) The top left panel shows a schematic illustration of the SSA substrate and TetO array located ∼15 kb from the HO cut site on chromosome III. The bottom left panel shows representative images of DIC merged with Venus-Rad1 and TetR-mRFP1 either before or after HO induction. Right panel shows percent co-localization of Venus-Rad1 with TetR-mRFP1 in wild-type or isoge