A tale of tails: insights into the coordination of 3′ end processing during homologous recombination

Eukaryotic genomes harbor a large number of homologous repeat sequences that are capable of recombining. Their potential to disrupt genome stability highlights the need to understand how homologous recombination processes are coordinated. The Saccharomyces cerevisiae Rad1–Rad10 endonuclease performs an essential role in recombination between repeated sequences, by processing 3′ single‐stranded intermediates formed during single‐strand annealing and gene conversion events. Several recent studies have focused on factors involved in Rad1–Rad10‐dependent removal of 3′ nonhomologous tails during homologous recombination, including Msh2–Msh3, Slx4, and the newly identified Saw1 protein. Together, this new work provides a model for how Rad1–Rad10‐dependent end processing is coordinated: Msh2–Msh3 stabilizes and prepares double‐strand/single‐strand junctions for Rad1–Rad10 cleavage, Saw1 recruits Rad1–Rad10 to 3′ tails, and Slx4 mediates crosstalk between the DNA damage checkpoint machinery and Rad1–Rad10.     

Microarray-based genetic screen defines SAW1, a gene required for Rad1/Rad10-dependent processing of recombination intermediates

Elimination of a double-strand break (DSB) flanked by direct repeat sequences is mediated by single-strand annealing (SSA), which relies on a distinct set of gene products involving recombination, mismatch repair, and nucleotide excision repair. Here, we screened for yeast mutants defective in SSA with a plasmid-based SSA assay coupled to a barcode microarray readout. The screen identified Yal027Wp/Saw1 (single-strand annealing weakened 1) and Slx4 besides other known SSA proteins. Saw1 interacts physically with Rad1/Rad10, Msh2/Msh3, and Rad52 proteins, and cells lacking SLX4 or SAW1 accumulate recombination intermediates blocked at the Rad1/Rad10-dependent 3' flap cleavage step. Slx4 and Saw1 also contribute to the integrity of ribosomal DNA arrays. Saw1 mutants that fail to interact with Rad1, but retain interaction with Rad52 and Msh2, are defective in 3' flap removal and SSA repair. Deletion of SAW1 abolished association of Rad1 at SSA intermediates in vivo. We propose that Saw1 targets Rad1/Rad10 to Rad52-coated recombination intermediates.     

SAW1 is increasingly required to recruit Rad10 as SSA flap-length increases from 20 to 50 bases in single-strand annealing in S. cerevisiae

SAW1 is required by the Rad1-Rad10 nuclease for efficient removal of 3′ non-homologous DNA ends (flaps) formed as intermediates during two modes of double-strand break repair in S. cerevisiae, single-strand annealing (SSA) and synthesis-dependent strand annealing (SDSA). Saw1 was shown in vitro to exhibit increasing affinity for flap DNAs as flap lengths varied from 0 to 40 deoxynucleotides (nt) with almost no binding observed when flaps were shorter than 10 nt. Accordingly, our prior in vivo fluorescence microscopy investigation showed that SAW1 was not required for recruitment of Rad10-YFP to DNA double-strand breaks (DSBs) when flaps were ～10 nt, but it was required when flaps were ～500 nt in G1 phase of the cell cycle. We were curious whether we would also observe an increased requirement of SAW1 for Rad10 recruitment in vivo as flaps varied from ～20 to 50 nt, as was shown in vitro. In this investigation, we utilized SSA substrates that generate 20, 30, and 50 nt flaps in vivo in fluorescence microscopy assays and determined that SAW1 becomes increasingly necessary for SSA starting at about ～20 nt and is completely required at ～50 nt. Quantitative PCR experiments corroborate these results by demonstrating that repair product formation decreases in the absence of SAW1 as flap length increases. Experiments with strains containing fluorescently labeled Saw1 (Saw1-CFP) show that Saw1 localizes with Rad10 at SSA foci and that about half of the foci containing Rad10 at DSBs do not contain Saw1. Colocalization patterns of Saw1-CFP are consistent regardless of the flap length of the substrate and are roughly similar in all phases of the cell cycle. Together, these data show that Saw1 becomes increasingly important for Rad1-Rad10 recruitment and SSA repair in the ～20–50 nt flap range, and Saw1 is present at repair sites even when not required and may depart the repair site ahead of Rad1-Rad10.     

Saccharomyces cerevisiae Mre11/Rad50/Xrs2 and Ku proteins regulate association of Exo1 and Dna2 with DNA breaks

Single‐stranded DNA constitutes an important early intermediate for homologous recombination and damage‐induced cell cycle checkpoint activation. In Saccharomyces cerevisiae, efficient double‐strand break (DSB) end resection requires several enzymes; Mre11/Rad50/Xrs2 (MRX) and Sae2 are implicated in the onset of 5′‐strand resection, whereas Sgs1/Top3/Rmi1 with Dna2 and Exo1 are involved in extensive resection. However, the molecular events leading to a switch from the MRX/Sae2‐dependent initiation to the Exo1‐ and Dna2‐dependent resection remain unclear. Here, we show that MRX recruits Dna2 nuclease to DSB ends. MRX also stimulates recruitment of Exo1 and antagonizes excess binding of the Ku complex to DSB ends. Using resection assay with purified enzymes in vitro, we found that Ku and MRX regulate the nuclease activity of Exo1 in an opposite way. Efficient loading of Dna2 and Exo1 requires neither Sae2 nor Mre11 nuclease activities. However, Mre11 nuclease activity is essential for resection in the absence of extensive resection enzymes. The results provide new insights into how MRX catalyses end resection and recombination initiation.     

Structural mechanism of ATP‐dependent DNA binding and DNA end bridging by eukaryotic Rad50

The Mre11–Rad50–Nbs1 (MRN) complex is a central factor in the repair of DNA double‐strand breaks (DSBs). The ATP‐dependent mechanisms of how MRN detects and endonucleolytically processes DNA ends for the repair by microhomology‐mediated end‐joining or further resection in homologous recombination are still unclear. Here, we report the crystal structures of the ATPγS‐bound dimer of the Rad50^NBD (nucleotide‐binding domain) from the thermophilic eukaryote Chaetomium thermophilum (Ct) in complex with either DNA or CtMre11^RBD (Rad50‐binding domain) along with small‐angle X‐ray scattering and cross‐linking studies. The structure and DNA binding motifs were validated by DNA binding experiments in vitro and mutational analyses in Saccharomyces cerevisiae in vivo. Our analyses provide a structural framework for the architecture of the eukaryotic Mre11–Rad50 complex. They show that a Rad50 dimer binds approximately 18 base pairs of DNA along the dimer interface in an ATP‐dependent fashion or bridges two DNA ends with a preference for 3′ overhangs. Finally, our results may provide a general framework for the interaction of ABC ATPase domains of the Rad50/SMC/RecN protein family with DNA.     

SAW1 is required for SDSA double-strand break repair in S. cerevisiae

SAW1, coding for Saw1, is required for single-strand annealing (SSA) DNA double-strand break (DSB) repair in Saccharomycescerevisiae. Saw1 physically associates with Rad1 and Rad52 and recruits the Rad1–Rad10 endonuclease. Herein we show by fluorescence microscopy that SAW1 is similarly required for recruitment of Rad10 to sites of Synthesis-Dependent Strand Annealing (SDSA) and associates with sites of SDSA repair in a manner temporally overlapped with Rad10. The magnitude of induction of colocalized Saw1-CFP/Rad10-YFP/DSB-RFP foci in SDSA is more dramatic in S and G2 phase cells than in M phase, consistent with the known mechanism of SDSA. We observed a substantial fraction of foci in which Rad10 was localized to the repair site without Saw1, but few DSB sites that contained Saw1 without Rad10. Together these data are consistent with a model in which Saw1 recruits Rad1–Rad10 to SDSA sites, possibly even binding as a protein–protein complex, but departs the repair site in advance of Rad1–Rad10.     

Requirement of Mismatch Repair Genes Msh2 and Msh3 in the Rad1-Rad10 Pathway of Mitotic Recombination in Saccharomyces Cerevisiae

The RAD1 and RAD10 genes of Saccharomyces cerevisiae are required for nucleotide excision repair and they also act in mitotic recombination. The Rad1-Rad10 complex has a single-stranded DNA endonuclease activity. Here, we show that the mismatch repair genes MSH2 and MSH3 function in mitotic recombination. For both his3 and his4 duplications, and for homologous integration of a linear DNA fragment into the genome, the msh3Δ mutation has an effect on recombination similar to that of the rad1Δ and rad10Δ mutations. The msh2Δ mutation also reduces the rate of recombination of the his3 duplication and lowers the incidence of homologous integration of a linear DNA fragment. Epistasis analyses indicate that MSH2 and MSH3 function in the RAD1-RAD10 recombination pathway, and studies presented here suggest an involvement of the RAD1-RAD10 pathway in reciprocal recombination. The possible roles of Msh2, Msh3, Rad1, and Rad10 proteins in genetic recombination are discussed. Coupling of mismatch binding proteins with the recombinational machinery could be important for ensuring genetic fidelity in the recombination process.     

A novel protein,Rsf1/Pxd1, is critical for the single‐strand annealing pathway of double‐strand break repair in Schizosaccharomyces pombe

The process of single‐strand annealing (SSA) repairs DNA double‐strand breaks that are flanked by direct repeat sequences through the coordinated actions of a series of proteins implicated in recombination, mismatch repair and nucleotide excision repair (NER). Many of the molecular and mechanistic insights gained in SSA repair have principally come from studies in the budding yeast Saccharomyces cerevisiae. However, there is little molecular understanding of the SSA pathway in the fission yeast Schizosaccharomyces pombe. To further our understanding of this important process, we established a new chromosome‐based SSA assay in fission yeast. Our genetic analyses showed that, although many homologous components participate in SSA repair in these species indicating that some evolutionary conservation, Saw1 and Slx4 are not principal agents in the SSA repair pathway in fission yeast. This is in marked contrast to the function of Saw1 and Slx4 in budding yeast. Additionally, a novel genus‐specific protein, Rsf1/Pxd1, physically interacts with Rad16, Swi10 and Saw1 in vitro and in vivo. We find that Rsf1/Pxd1 is not required for NER and demonstrate that, in fission yeast, Rsf1/Pxd1, but not Saw1, plays a critical role in SSA recombination.     

Escape of Sgs1 from Rad9 inhibition reduces the requirement for Sae2 and functional MRX in DNA end resection

Homologous recombination requires nucleolytic degradation (resection) of DNA double‐strand break (DSB) ends. In Saccharomyces cerevisiae, the MRX complex and Sae2 are involved in the onset of DSB resection, whereas extensive resection requires Exo1 and the concerted action of Dna2 and Sgs1. Here, we show that the checkpoint protein Rad9 limits the action of Sgs1/Dna2 in DSB resection by inhibiting Sgs1 binding/persistence at the DSB ends. When inhibition by Rad9 is abolished by the Sgs1‐ss mutant variant or by deletion of RAD9, the requirement for Sae2 and functional MRX in DSB resection is reduced. These results provide new insights into how early and long‐range resection is coordinated.     

Mec1/ATR regulates the generation of single‐stranded DNA that attenuates Tel1/ATM signaling at DNA ends

Tel1/ATM and Mec1/ATR checkpoint kinases are activated by DNA double‐strand breaks (DSBs). Mec1/ATR recruitment to DSBs requires the formation of RPA‐coated single‐stranded DNA (ssDNA), which arises from 5′–3′ nucleolytic degradation (resection) of DNA ends. Here, we show that Saccharomyces cerevisiae Mec1 regulates resection of the DSB ends. The lack of Mec1 accelerates resection and reduces the loading to DSBs of the checkpoint protein Rad9, which is known to inhibit ssDNA generation. Extensive resection is instead inhibited by the Mec1‐ad mutant variant that increases the recruitment near the DSB of Rad9, which in turn blocks DSB resection by both Rad53‐dependent and Rad53‐independent mechanisms. The mec1‐ad resection defect leads to prolonged persistence at DSBs of the MRX complex that causes unscheduled Tel1 activation, which in turn impairs checkpoint switch off. Thus, Mec1 regulates the generation of ssDNA at DSBs, and this control is important to coordinate Mec1 and Tel1 signaling activities at these breaks.     

Yeast DNA recombination and repair proteins Rad 1 and Radio constitute a complex in vivo mediated by localized hydrophobic domains

The Saccharomyces cerevisiae Rad 1 and Rad 10 proteins are required for damage-specific incision during nucleotide excision repair and also for certain mitotic recombination events between repeated sequences. Previously we have demonstrated that Rad1 and Rad10 form a specific complex in vitro. Using the ‘two-hybrid’ genetic assay system we now report that Rad1 and Rad10 proteins are subunits of a specific complex in the cell nucleus. The Rad10-binding domain of Rad1 protein maps to a localized region between amino acids 809–997. The Rad1 -binding domain of Radio protein maps between amino acids 90–210. These domains are evolutionarily conserved and are hydrophobic in character. Although significant homology exists between Rad10 and the human-DNA-repair protein Ercc1 in this region, we were unable to detect any interaction between Ercc1 and Rad1 proteins. We conclude that Rad1 and Rad10 operate in DNA repair and mitotic recombination as a constitutive complex.     

Multiple Regulation of Rad51-Mediated Homologous Recombination by Fission Yeast Fbh1

Fbh1, an F-box helicase related to bacterial UvrD, has been proposed to modulate homologous recombination in fission yeast. We provide several lines of evidence for such modulation. Fbh1, but not the related helicases Srs2 and Rqh1, suppressed the formation of crossover recombinants from single HO-induced DNA double-strand breaks. Purified Fbh1 in complex with Skp1 (Fbh1-Skp1 complex) inhibited Rad51-driven DNA strand exchange by disrupting Rad51 nucleoprotein filaments in an ATP-dependent manner; this disruption was alleviated by the Swi5-Sfr1 complex, an auxiliary activator of Rad51. In addition, the reconstituted SCF^Fbh1 complex, composed of purified Fbh1-Skp1 and Pcu1-Rbx1, displayed ubiquitin-ligase E3 activity toward Rad51. Furthermore, Fbh1 reduced the protein level of Rad51 in stationary phase in an F-box-dependent, but not in a helicase domain-independent manner. These results suggest that Fbh1 negatively regulates Rad51-mediated homologous recombination via its two putative, unrelated activities, namely DNA unwinding/translocation and ubiquitin ligation. In addition to its anti-recombinase activity, we tentatively suggest that Fbh1 might also have a pro-recombination role in vivo, because the Fbh1-Skp1 complex stimulated Rad51-mediated strand exchange in vitro after strand exchange had been initiated.     

Synergistic action of the Saccharomyces cerevisiae homologous recombination factors Rad54 and Rad51 in chromatin remodeling

Rad54, a member of the Swi2/Snf2 protein family, works in concert with the RecA-like recombinase Rad51 during the early and late stages of homologous recombination. Rad51 markedly enhances the activities of Rad54, including the induction of topological changes in DNA and the remodeling of chromatin structure. Reciprocally, Rad54 promotes Rad51-mediated DNA strand invasion with either naked or chromatinized DNA. Here, using various Saccharomyces cerevisiae rad51 and rad54 mutant proteins, mechanistic aspects of Rad54/Rad51-mediated chromatin remodeling are defined. Disruption of the Rad51-Rad54 complex leads to a marked attenuation of chromatin remodeling activity. Moreover, we present evidence that assembly of the Rad51 presynaptic filament represents an obligatory step in the enhancement of the chromatin remodeling reaction. Interestingly, we find a specific interaction of the N-terminal tail of histone H3 with Rad54 and show that the H3 tail interaction domain resides within the amino terminus of Rad54. These results suggest that Rad54-mediated chromatin remodeling coincides with DNA homology search by the Rad51 presynaptic filament and that this process is facilitated by an interaction of Rad54 with histone H3.     

Mgm101: A double-duty Rad52-like protein

Mgm101 has well-characterized activity for the repair and replication of the mitochondrial genome. Recent work has demonstrated a further role for Mgm101 in nuclear DNA metabolism, contributing to an S-phase specific DNA interstrand cross-link repair pathway that acts redundantly with a pathway controlled by Pso2 exonuclease. Due to involvement of FANCM, FANCJ and FANCP homologues (Mph1, Chl1 and Slx4), this pathway has been described as a Fanconi anemia-like pathway. In this pathway, Mgm101 physically interacts with the DNA helicase Mph1 and the MutSα (Msh2/Msh6) heterodimer, but its precise role is yet to be elucidated. Data presented here suggests that Mgm101 functionally overlaps with Rad52, supporting previous suggestions that, based on protein structure and biochemical properties, Mgm101 and Rad52 belong to a family of proteins with similar function. In addition, our data shows that this overlap extends to the function of both proteins at telomeres, where Mgm101 is required for telomere elongation during chromosome replication in rad52 defective cells. We hypothesize that Mgm101 could, in Rad52-like manner, preferentially bind single-stranded DNAs (such as at stalled replication forks, broken chromosomes and natural chromosome ends), stabilize them and mediate single-strand annealing-like homologous recombination event to prevent them from converting into toxic structures.     

Multiple pathways promote short-sequence recombination in Saccharomyces cerevisiae

In the budding yeast Saccharomyces cerevisiae, null alleles of several DNA repair and recombination genes confer defects in recombination that grow more severe with decreasing sequence length, indicating that they are required for short-sequence recombination (SSR). RAD1 and RAD10, which encode the subunits of the structure-specific endonuclease Rad1/10, are critical for SSR. MRE11, RAD50, and XRS2, which encode the subunits of M/R/X, another complex with nuclease activity, are also crucially important. Genetic evidence suggests that Rad1/10 and M/R/X act on the same class of substrates during SSR. MSH2 and MSH3, which encode subunits of Msh2/3, a complex active during mismatch repair and recombination, are also important for SSR but play a more restricted role. Additional evidence suggests that SSR is distinct from nonhomologous end joining and is superimposed upon basal homologous recombination.     

Opposing roles for DNA structure-specific proteins Rad1, Msh2, Msh3, and Sgs1 in yeast gene targeting

Targeted gene replacement (TGR) in yeast and mammalian cells is initiated by the two free ends of the linear targeting molecule, which invade their respective homologous sequences in the chromosome, leading to replacement of the targeted locus with a selectable gene from the targeting DNA. To study the postinvasion steps in recombination, we examined the effects of DNA structure-specific proteins on TGR frequency and heteroduplex DNA formation. In strains deleted of RAD1, MSH2, or MSH3, we find that the frequency of TGR is reduced and the mechanism of TGR is altered while the reverse is true for deletion of SGS1, suggesting that Rad1 and Msh2:Msh3 facilitate TGR while Sgs1 opposes it. The altered mechanism of TGR in the absence of Msh2:Msh3 and Rad1 reveals a separate role for these proteins in suppressing an alternate gene replacement pathway in which incorporation of both homology regions from a single strand of targeting DNA into heteroduplex with the targeted locus creates a mismatch between the selectable gene on the targeting DNA and the targeted gene in the chromosome.     

Rrp1 translocase and ubiquitin ligase activities restrict the genome destabilising effects of Rad51 in fission yeast

Rad51 is the key protein in homologous recombination that plays important roles during DNA replication and repair. Auxiliary factors regulate Rad51 activity to facilitate productive recombination, and prevent inappropriate, untimely or excessive events, which could lead to genome instability. Previous genetic analyses identified a function for Rrp1 (a member of the Rad5/16-like group of SWI2/SNF2 translocases) in modulating Rad51 function, shared with the Rad51 mediator Swi5-Sfr1 and the Srs2 anti-recombinase. Here, we show that Rrp1 overproduction alleviates the toxicity associated with excessive Rad51 levels in a manner dependent on Rrp1 ATPase domain. Purified Rrp1 binds to DNA and has a DNA-dependent ATPase activity. Importantly, Rrp1 directly interacts with Rad51 and removes it from double-stranded DNA, confirming that Rrp1 is a translocase capable of modulating Rad51 function. Rrp1 affects Rad51 binding at centromeres. Additionally, we demonstrate in vivo and in vitro that Rrp1 possesses E3 ubiquitin ligase activity with Rad51 as a substrate, suggesting that Rrp1 regulates Rad51 in a multi-tiered fashion.     

Regulation of mitotic homeologous recombination in yeast. Functions of mismatch repair and nucleotide excision repair genes

The Saccharomyces cerevisiae homologs of the bacterial mismatch repair proteins MutS and MutL correct replication errors and prevent recombination between homeologous (nonidentical) sequences. Previously, we demonstrated that Msh2p, Msh3p, and Pms1p regulate recombination between 91% identical inverted repeats, and here use the same substrates to show that Mlh1p and Msh6p have important antirecombination roles. In addition, substrates containing defined types of mismatches (base-base mismatches; 1-, 4-, or 12-nt insertion/deletion loops; or 18-nt palindromes) were used to examine recognition of these mismatches in mitotic recombination intermediates. Msh2p was required for recognition of all types of mismatches, whereas Msh6p recognized only base-base mismatches and 1-nt insertion/deletion loops. Msh3p was involved in recognition of the palindrome and all loops, but also had an unexpected antirecombination role when the potential heteroduplex contained only base-base mismatches. In contrast to their similar antimutator roles, Pms1p consistently inhibited recombination to a lesser degree than did Msh2p. In addition to the yeast MutS and MutL homologs, the exonuclease Exo1p and the nucleotide excision repair proteins Rad1p and Rad10p were found to have roles in inhibiting recombination between mismatched substrates.     

ATP binding and hydrolysis by Saccharomyces cerevisiae Msh2–Msh3 are differentially modulated by mismatch and double-strand break repair DNA substrates

In Saccharomyces cerevisiae, Msh2–Msh3-mediated mismatch repair (MMR) recognizes and targets insertion/deletion loops for repair. Msh2–Msh3 is also required for 3′ non-homologous tail removal (3′NHTR) in double-strand break repair. In both pathways, Msh2–Msh3 binds double-strand/single-strand junctions and initiates repair in an ATP-dependent manner. However, we recently demonstrated that the two pathways have distinct requirements with respect to Msh2–Msh3 activities. We identified a set of aromatic residues in the nucleotide binding pocket (FLY motif) of Msh3 that, when mutated, disrupted MMR, but left 3′NHTR largely intact. One of these mutations, msh3Y942A, was predicted to disrupt the nucleotide sandwich and allow altered positioning of ATP within the pocket. To develop a mechanistic understanding of the differential requirements for ATP binding and/or hydrolysis in the two pathways, we characterized Msh2–Msh3 and Msh2–msh3Y942A ATP binding and hydrolysis activities in the presence of MMR and 3′NHTR DNA substrates. We observed distinct, substrate-dependent ATP hydrolysis and nucleotide turnover by Msh2–Msh3, indicating that the MMR and 3′NHTR DNA substrates differentially modify the ATP binding/hydrolysis activities of Msh2–Msh3. Msh2–msh3Y942A retained the ability to bind DNA and ATP but exhibited altered ATP hydrolysis and nucleotide turnover. We propose that both ATP and structure-specific repair substrates cooperate to direct Msh2–Msh3-mediated repair and suggest an explanation for the msh3Y942A separation-of-function phenotype.     

Association of the Rad9–Rad1–Hus1 checkpoint clamp with MYH DNA glycosylase and DNA

Cell cycle checkpoints provide surveillance mechanisms to activate the DNA damage response, thus preserving genomic integrity. The heterotrimeric Rad9–Rad1–Hus1 (9–1–1) clamp is a DNA damage response sensor and can be loaded onto DNA. 9–1–1 is involved in base excision repair (BER) by interacting with nearly every enzyme in BER. Here, we show that individual 9–1–1 components play distinct roles in BER directed by MYH DNA glycosylase. Analyses of Hus1 deletion mutants revealed that the interdomain connecting loop (residues 134–155) is a key determinant of MYH binding. Both the N-(residues 1–146) and C-terminal (residues 147–280) halves of Hus1, which share structural similarity, can interact with and stimulate MYH. The Hus1^K136A mutant retains physical interaction with MYH but cannot stimulate MYH glycosylase activity. The N-terminal domain, but not the C-terminal half of Hus1 can also bind DNA with moderate affinity. Intact Rad9 expressed in bacteria binds to and stimulates MYH weakly. However, Rad9¹⁻²⁶⁶ (C-terminal truncated Rad9) can stimulate MYH activity and bind DNA with high affinity, close to that displayed by heterotrimeric 9¹⁻²⁶⁶–1–1 complexes. Conversely, Rad1 has minimal roles in stimulating MYH activity or binding to DNA. Finally, we show that preferential recruitment of 9¹⁻²⁶⁶–1–1 to 5′-recessed DNA substrates is an intrinsic property of this complex and is dependent on complex formation. Together, our findings provide a mechanistic rationale for unique contributions by individual 9–1–1 subunits to MYH-directed BER based on subunit asymmetry in protein–protein interactions and DNA binding events.