Release of Ku and MRN from DNA ends by Mre11 nuclease activity and Ctp1 is required for homologous recombination repair of double-strand breaks

The multifunctional Mre11-Rad50-Nbs1 (MRN) protein complex recruits ATM/Tel1 checkpoint kinase and CtIP/Ctp1 homologous recombination (HR) repair factor to double-strand breaks (DSBs). HR repair commences with the 5'-to-3' resection of DNA ends, generating 3' single-strand DNA (ssDNA) overhangs that bind Replication Protein A (RPA) complex, followed by Rad51 recombinase. In Saccharomyces cerevisiae, the Mre11-Rad50-Xrs2 (MRX) complex is critical for DSB resection, although the enigmatic ssDNA endonuclease activity of Mre11 and the DNA-end processing factor Sae2 (CtIP/Ctp1 ortholog) are largely unnecessary unless the resection activities of Exo1 and Sgs1-Dna2 are also eliminated. Mre11 nuclease activity and Ctp1/CtIP are essential for DSB repair in Schizosaccharomyces pombe and mammals. To investigate DNA end resection in Schizo. pombe, we adapted an assay that directly measures ssDNA formation at a defined DSB. We found that Mre11 and Ctp1 are essential for the efficient initiation of resection, consistent with their equally crucial roles in DSB repair. Exo1 is largely responsible for extended resection up to 3.1 kb from a DSB, with an activity dependent on Rqh1 (Sgs1) DNA helicase having a minor role. Despite its critical function in DSB repair, Mre11 nuclease activity is not required for resection in fission yeast. However, Mre11 nuclease and Ctp1 are required to disassociate the MRN complex and the Ku70-Ku80 nonhomologous end-joining (NHEJ) complex from DSBs, which is required for efficient RPA localization. Eliminating Ku makes Mre11 nuclease activity dispensable for MRN disassociation and RPA localization, while improving repair of a one-ended DSB formed by replication fork collapse. From these data we propose that release of the MRN complex and Ku from DNA ends by Mre11 nuclease activity and Ctp1 is a critical step required to expose ssDNA for RPA localization and ensuing HR repair.     

Synthetic viability genomic screening defines Sae2 function in DNA repair

DNA double-strand break (DSB) repair by homologous recombination (HR) requires 3′ single-stranded DNA (ssDNA) generation by 5′ DNA-end resection. During meiosis, yeast Sae2 cooperates with the nuclease Mre11 to remove covalently bound Spo11 from DSB termini, allowing resection and HR to ensue. Mitotic roles of Sae2 and Mre11 nuclease have remained enigmatic, however, since cells lacking these display modest resection defects but marked DNA damage hypersensitivities. By combining classic genetic suppressor screening with high-throughput DNA sequencing, we identify Mre11 mutations that strongly suppress DNA damage sensitivities of sae2Δ cells. By assessing the impacts of these mutations at the cellular, biochemical and structural levels, we propose that, in addition to promoting resection, a crucial role for Sae2 and Mre11 nuclease activity in mitotic DSB repair is to facilitate the removal of Mre11 from ssDNA associated with DSB ends. Thus, without Sae2 or Mre11 nuclease activity, Mre11 bound to partly processed DSBs impairs strand invasion and HR.     

Association of Mre11p with double-strand break sites during yeast meiosis

The repair of DNA double-strand breaks (DSBs) requires the activity of the Mre11/Rad50/Xrs2(Nbs1) complex. In Saccharomyces cerevisiae, this complex is required for both the initiation of meiotic recombination by Spo11p-catalyzed programmed DSBs and for break end resection, which is necessary for repair by homologous recombination. We report that Mre11p transiently associates with the chromatin of Spo11-dependent DSB regions throughout the genome. Mutant analyses show that Mre11p binding requires the function of all genes required for DSB formation, with the exception of RAD50. However, Mre11p binding does not require DSB formation itself, since Mre11p transiently associates with DSB regions in the catalysis-negative mutant spo11-Y135F. Mre11p release from chromatin is blocked in mutants that accumulate unresected DSBs. We propose that Mre11p is a component of a pre-DSB complex that assembles on the DSB sites, thus ensuring a tight coupling between DSB formation by Spo11p and the processing of break ends.     

Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends

Formation of single-strand DNA (ssDNA) tails at a double-strand break (DSB) is a key step in homologous recombination and DNA-damage signaling. The enzyme(s) producing ssDNA at DSBs in eukaryotes remain unknown. We monitored 5'-strand resection at inducible DSB ends in yeast and identified proteins required for two stages of resection: initiation and long-range 5'-strand resection. We show that the Mre11-Rad50-Xrs2 complex (MRX) initiates 5' degradation, whereas Sgs1 and Dna2 degrade 5' strands exposing long 3' strands. Deletion of SGS1 or DNA2 reduces resection and DSB repair by single-strand annealing between distant repeats while the remaining long-range resection activity depends on the exonuclease Exo1. In exo1Deltasgs1Delta double mutants, the MRX complex together with Sae2 nuclease generate, in a stepwise manner, only few hundred nucleotides of ssDNA at the break, resulting in inefficient gene conversion and G2/M damage checkpoint arrest. These results provide important insights into the early steps of DSB repair in eukaryotes.     

Mechanism of RecO recruitment to DNA by single-stranded DNA binding protein

RecO is a recombination mediator protein (RMP) important for homologous recombination, replication repair and DNA annealing in bacteria. In all pathways, the single-stranded (ss) DNA binding protein, SSB, plays an inhibitory role by protecting ssDNA from annealing and recombinase binding. Conversely, SSB may stimulate each reaction through direct interaction with RecO. We present a crystal structure of Escherichia coli RecO bound to the conserved SSB C-terminus (SSB-Ct). SSB-Ct binds the hydrophobic pocket of RecO in a conformation similar to that observed in the ExoI/SSB-Ct complex. Hydrophobic interactions facilitate binding of SSB-Ct to RecO and RecO/RecR complex in both low and moderate ionic strength solutions. In contrast, RecO interaction with DNA is inhibited by an elevated salt concentration. The SSB mutant lacking SSB-Ct also inhibits RecO-mediated DNA annealing activity in a salt-dependent manner. Neither RecO nor RecOR dissociates SSB from ssDNA. Therefore, in E. coli, SSB recruits RMPs to ssDNA through SSB-Ct, and RMPs are likely to alter the conformation of SSB-bound ssDNA without SSB dissociation to initiate annealing or recombination. Intriguingly, Deinococcus radiodurans RecO does not bind SSB-Ct and weakly interacts with the peptide in the presence of RecR, suggesting the diverse mechanisms of DNA repair pathways mediated by RecO in different organisms.     

hMre11 and hRad50 nuclear foci are induced during the normal cellular response to DNA double-strand breaks.

We previously identified a conserved multiprotein complex that includes hMre11 and hRad50. In this study, we used immunofluorescence to investigate the role of this complex in DNA double-strand break (DSB) repair. hMre11 and hRad50 form discrete nuclear foci in response to treatment with DSB-inducing agents but not in response to UV irradiation. hMre11 and hRad50 foci colocalize after treatment with ionizing radiation and are distinct from those of the DSB repair protein, hRad51. Our data indicate that an irradiated cell is competent to form either hMre11-hRad50 foci or hRad51 foci, but not both. The multiplicity of hMre11 and hRad50 foci is much higher in the DSB repair-deficient cell line 180BR than in repair-proficient cells. hMre11-hRad50 focus formation is markedly reduced in cells derived from ataxia-telangiectasia patients, whereas hRad51 focus formation is markedly increased. These experiments support genetic evidence from Saccharomyces cerevisiae indicating that Mre11-Rad50 have roles distinct from that of Rad51 in DSB repair. Further, these data indicate that hMre11-hRad50 foci form in response to DNA DSBs and are dependent upon a DNA damage-induced signaling pathway.     

Mre11 dimers coordinate DNA end bridging and nuclease processing in double-strand-break repair

Mre11 forms the core of the multifunctional Mre11-Rad50-Nbs1 (MRN) complex that detects DNA double-strand breaks (DSBs), activates the ATM checkpoint kinase, and initiates homologous recombination (HR) repair of DSBs. To define the roles of Mre11 in both DNA bridging and nucleolytic processing during initiation of DSB repair, we combined small-angle X-ray scattering (SAXS) and crystal structures of Pyrococcus furiosus Mre11 dimers bound to DNA with mutational analyses of fission yeast Mre11. The Mre11 dimer adopts a four-lobed U-shaped structure that is critical for proper MRN complex assembly and for binding and aligning DNA ends. Further, mutations blocking Mre11 endonuclease activity impair cell survival after DSB induction without compromising MRN complex assembly or Mre11-dependant recruitment of Ctp1, an HR factor, to DSBs. These results show how Mre11 dimerization and nuclease activities initiate repair of DSBs and collapsed replication forks, as well as provide a molecular foundation for understanding cancer-causing Mre11 mutations in ataxia telangiectasia-like disorder (ATLD).     

The Mre11-Rad50-Xrs2 Protein Complex Facilitates Homologous Recombination-Based Double-Strand Break Repair in Saccharomyces cerevisiae

Saccharomyces cerevisiae mre11Delta mutants are profoundly deficient in double-strand break (DSB) repair, indicating that the Mre11-Rad50-Xrs2 protein complex plays a central role in the cellular response to DNA DSBs. In this study, we examined the role of the complex in homologous recombination, the primary mode of DSB repair in yeast. We measured survival in synchronous cultures following irradiation and scored sister chromatid and interhomologue recombination genetically. mre11Delta strains were profoundly sensitive to ionizing radiation (IR) throughout the cell cycle. Mutant strains exhibited decreased frequencies of IR-induced sister chromatid and interhomologue recombination, indicating a general deficiency in homologous recombination-based DSB repair. Since a nuclease-deficient mre11 mutant was not impaired in these assays, it appears that the role of the S. cerevisiae Mre11-Rad50-Xrs2 protein complex in facilitating homologous recombination is independent of its nuclease activities.     

Stimulation of Dmc1-mediated DNA strand exchange by the human Rad54B protein

The process of homologous recombination is indispensable for both meiotic and mitotic cell division, and is one of the major pathways for double-strand break (DSB) repair. The human Rad54B protein, which belongs to the SWI2/SNF2 protein family, plays a role in homologous recombination, and may function with the Dmc1 recombinase, a meiosis-specific Rad51 homolog. In the present study, we found that Rad54B enhanced the DNA strand-exchange activity of Dmc1 by stabilizing the Dmc1–single-stranded DNA (ssDNA) complex. Therefore, Rad54B may stimulate the Dmc1-mediated DNA strand exchange by stabilizing the nucleoprotein filament, which is formed on the ssDNA tails produced at DSB sites during homologous recombination.     

Mammalian X-ray-sensitive mutants which are defective in non-homologous (illegitimate) DNA double-strand break repair

In all organisms multiple pathways to repair DNA double-strand breaks (DSB) have been identified. In mammalian cells DSB are repaired by two distinct pathways, homologous and non-homologous (illegitimate) recombination. X-ray-sensitive mutants have provided a tool for the identification and understanding of the illegitimate recombination pathway in mammalian cells. Two (sub-)pathways can be distinguished, the first mediated by DNA-PK-dependent protein kinase (DNA-PK), and the second directed by the hMre11/hRad50 complex. A variety of mutants impaired in DSB repair by illegitimate recombination, with mutations in Ku, DNA-PKcs, XRCC4 or nibrin, have been described. Herein, the characterization of these mutants with respect to the impaired cellular function and the molecular defect is provided. Further studies on these mutants, as well as on new mutants impaired in as-of-yet unidentified pathways, should be helpful to a better understanding of DSB repair and of the processes leading to genome instability and cancer.     

RecO Protein Initiates DNA Recombination and Strand Annealing through Two Alternative DNA Binding Mechanisms

Recombination mediator proteins (RMPs) are important for genome stability in all organisms. Several RMPs support two alternative reactions: initiation of homologous recombination and DNA annealing. We examined mechanisms of RMPs in both reactions with Mycobacterium smegmatis RecO (MsRecO) and demonstrated that MsRecO interacts with ssDNA by two distinct mechanisms. Zinc stimulates MsRecO binding to ssDNA during annealing, whereas the recombination function is zinc-independent and is regulated by interaction with MsRecR. Thus, different structural motifs or conformations of MsRecO are responsible for interaction with ssDNA during annealing and recombination. Neither annealing nor recombinase loading depends on MsRecO interaction with the conserved C-terminal tail of single-stranded (ss) DNA-binding protein (SSB), which is known to bind Escherichia coli RecO. However, similarly to E. coli proteins, MsRecO and MsRecOR do not dismiss SSB from ssDNA, suggesting that RMPs form a complex with SSB-ssDNA even in the absence of binding to the major protein interaction motif. We propose that alternative conformations of such complexes define the mechanism by which RMPs initiate the repair of stalled replication and support two different functions during recombinational repair of DNA breaks.     

Conditional gene targeted deletion by Cre recombinase demonstrates the requirement for the double-strand break repair Mre11 protein in murine embryonic stem cells.

Repair of DNA damage resulting in double-strand breaks (DSBs) is controlled by gene products executing homologous recombination or end-joining pathways. The MRE11 gene has previously been implicated in DSB repair in the yeast Saccharomyces cerevisiae . Here we have developed a methodology to study the roles of the murine Mre11 homolog in pluripotent embryonic stem cells. Using a gene targeting approach, a triple LoxP site cassette was inserted into a region of MRE11 genomic DNA flanking conserved phosphodiesterase motifs. The addition of Cre recombinase activity promotes deletions of three types that can be scored. We find that deletion at phosphodiesterase motif III encoded in the N-terminus of Mre11 is acheived in the presence of a wild-type MRE11 allele. However, when the wild-type MRE11 allele is inactivated by gene targeted insertion of a neo marker, only Cre recombination events that allow expression of wild-type Mre11 protein are observed. Therefore, Mre11 is required for normal cell proliferation. This methodology introduces a means to study important regions of essential genes in cell culture models.     

Distinct roles of two separable in vitro activities of yeast Mre11 in mitotic and meiotic recombination.

In Saccharomyces cerevisiae, Mre11 protein is involved in both double-strand DNA break (DSB) repair and meiotic DSB formation. Here, we report the correlation of nuclease and DNA-binding activities of Mre11 with its functions in DNA repair and meiotic DSB formation. Purified Mre11 bound to DNA efficiently and was shown to have Mn2+-dependent nuclease activities. A point mutation in the N-terminal phosphoesterase motif (Mre11D16A) resulted in the abolition of nuclease activities but had no significant effect on DNA binding. The wild-type level of nuclease activity was detected in a C-terminal truncated protein (Mre11DeltaC49), although it had reduced DNA-binding activity. Phenotypes of the corresponding mutations were also analyzed. The mre11D16A mutation conferred methyl methanesulfonate-sensitivity to mitotic cells and caused the accumulation of unprocessed meiotic DSBs. The mre11DeltaC49 mutant exhibited almost wild-type phenotypes in mitosis. However, in meiosis, no DSB formation could be detected and an aberrant chromatin configuration was observed at DSB sites in the mre11DeltaC49 mutant. These results indicate that Mre11 has two separable functional domains: the N-terminal nuclease domain required for DSB repair, and the C-terminal dsDNA-binding domain essential to its meiotic functions such as chromatin modification and DSB formation. Keywords: DNA binding/double-strand break repair/DSB formation/Mre11/nuclease     

Excess single-stranded DNA inhibits meiotic double-strand break repair

During meiosis, self-inflicted DNA double-strand breaks (DSBs) are created by the protein Spo11 and repaired by homologous recombination leading to gene conversions and crossovers. Crossover formation is vital for the segregation of homologous chromosomes during the first meiotic division and requires the RecA orthologue, Dmc1.We analyzed repair during meiosis of site-specific DSBs created by another nuclease, VMA1-derived endonuclease (VDE), in cells lacking Dmc1 strand-exchange protein. Turnover and resection of the VDE-DSBs was assessed in two different reporter cassettes that can repair using flanking direct repeat sequences, thereby obviating the need for a Dmc1-dependent DNA strand invasion step. Access of the single-strand binding complex replication protein A, which is normally used in all modes of DSB repair, was checked in chromatin immunoprecipitation experiments, using antibody against Rfa1. Repair of the VDE-DSBs was severely inhibited in dmc1Δ cells, a defect that was associated with a reduction in the long tract resection required to initiate single-strand annealing between the flanking repeat sequences. Mutants that either reduce Spo11-DSB formation or abolish resection at Spo11-DSBs rescued the repair block. We also found that a replication protein A component, Rfa1, does not accumulate to expected levels at unrepaired single-stranded DNA (ssDNA) in dmc1Δ cells. The requirement of Dmc1 for VDE-DSB repair using flanking repeats appears to be caused by the accumulation of large quantities of ssDNA that accumulate at Spo11-DSBs when Dmc1 is absent. We propose that these resected DSBs sequester both resection machinery and ssDNA binding proteins, which in wild-type cells would normally be recycled as Spo11-DSBs repair. The implication is that repair proteins are in limited supply, and this could reflect an underlying mechanism for regulating DSB repair in wild-type cells, providing protection from potentially harmful effects of overabundant repair proteins.     

Role of Saccharomyces Single-Stranded DNA-Binding Protein RPA in the Strand Invasion Step of Double-Strand Break Repair

The single-stranded DNA (ssDNA)-binding protein replication protein A (RPA) is essential for both DNA replication and recombination. Chromatin immunoprecipitation techniques were used to visualize the kinetics and extent of RPA binding following induction of a double-strand break (DSB) and during its repair by homologous recombination in yeast. RPA assembles at the HO endonuclease-cut MAT locus simultaneously with the appearance of the DSB, and binding spreads away from the DSB as 5′ to 3′ exonuclease activity creates more ssDNA. RPA binding precedes binding of the Rad51 recombination protein. The extent of RPA binding is greater when Rad51 is absent, supporting the idea that Rad51 displaces RPA from ssDNA. RPA plays an important role during RAD51-mediated strand invasion of the MAT ssDNA into the donor sequence HML. The replication-proficient but recombination-defective rfa1-t11 (K45E) mutation in the large subunit of RPA is normal in facilitating Rad51 filament formation on ssDNA, but is unable to achieve synapsis between MAT and HML. Thus, RPA appears to play a role in strand invasion as well as in facilitating Rad51 binding to ssDNA, possibly by stabilizing the displaced ssDNA.     

Role of Saccharomyces single-stranded DNA-binding protein RPA in the strand invasion step of double-strand break repair

The single-stranded DNA (ssDNA)-binding protein replication protein A (RPA) is essential for both DNA replication and recombination. Chromatin immunoprecipitation techniques were used to visualize the kinetics and extent of RPA binding following induction of a double-strand break (DSB) and during its repair by homologous recombination in yeast. RPA assembles at the HO endonuclease-cut MAT locus simultaneously with the appearance of the DSB, and binding spreads away from the DSB as 5′ to 3′ exonuclease activity creates more ssDNA. RPA binding precedes binding of the Rad51 recombination protein. The extent of RPA binding is greater when Rad51 is absent, supporting the idea that Rad51 displaces RPA from ssDNA. RPA plays an important role during RAD51-mediated strand invasion of the MAT ssDNA into the donor sequence HML. The replication-proficient but recombination-defective rfa1-t11 (K45E) mutation in the large subunit of RPA is normal in facilitating Rad51 filament formation on ssDNA, but is unable to achieve synapsis between MAT and HML. Thus, RPA appears to play a role in strand invasion as well as in facilitating Rad51 binding to ssDNA, possibly by stabilizing the displaced ssDNA.     

Bcl2 inhibits recruitment of Mre11 complex to DNA double-strand breaks in response to high-linear energy transfer radiation

High-linear energy transfer ionizing radiation, derived from high charge (Z) and energy (E) (HZE) particles, induces clustered/complex DNA double-strand breaks (DSBs) that include small DNA fragments, which are not repaired by the non-homologous end-joining (NHEJ) pathway. The homologous recombination (HR) DNA repair pathway plays a major role in repairing DSBs induced by HZE particles. The Mre11 complex (Mre11/Rad50/NBS1)-mediated resection of DSB ends is a required step in preparing for DSB repair via the HR DNA repair pathway. Here we found that expression of Bcl2 results in decreased HR activity and retards the repair of DSBs induced by HZE particles (i.e. ⁵⁶iron and ²⁸silicon) by inhibiting Mre11 complex activity. Exposure of cells to ⁵⁶iron or ²⁸silicon promotes Bcl2 to interact with Mre11 via the BH1 and BH4 domains. Purified Bcl2 protein directly suppresses Mre11 complex-mediated DNA resection in vitro. Expression of Bcl2 reduces the ability of Mre11 to bind DNA following exposure of cells to HZE particles. Our findings suggest that, after cellular exposure to HZE particles, Bcl2 may inhibit Mre11 complex-mediated DNA resection leading to suppression of the HR-mediated DSB repair in surviving cells, which may potentially contribute to tumor development.     

Human Ku70/80 protein blocks exonuclease 1-mediated DNA resection in the presence of human Mre11 or Mre11/Rad50 protein complex

DNA double strand breaks (DSB) are repaired by nonhomologous end-joining (NHEJ) or homologous recombination (HR). Recent genetic data in yeast shows that the choice between these two pathways for the repair of DSBs is via competition between the NHEJ protein, Ku, and the HR protein, Mre11/Rad50/Xrs2 (MRX) complex. To study the interrelationship between human Ku and Mre11 or Mre11/Rad50 (MR), we established an in vitro DNA end resection system using a forked model dsDNA substrate and purified human Ku70/80, Mre11, Mre11/Rad50, and exonuclease 1 (Exo1). Our study shows that the addition of Ku70/80 blocks Exo1-mediated DNA end resection of the forked dsDNA substrate. Although human Mre11 and MR bind to the forked double strand DNA, they could not compete with Ku for DNA ends or actively mediate the displacement of Ku from the DNA end either physically or via its exonuclease or endonuclease activity. Our in vitro studies show that Ku can block DNA resection and suggest that Ku must be actively displaced for DNA end processing to occur and is more complicated than the competition model established in yeast.     

Unwinding of synthetic replication and recombination substrates by Srs2

The budding yeast Srs2 protein possesses 3′ to 5′ DNA helicase activity and channels untimely recombination to post-replication repair by removing Rad51 from ssDNA. However, it also promotes recombination via a synthesis-dependent strand-annealing pathway (SDSA). Furthermore, at the replication fork, Srs2 is required for fork progression and prevents the instability of trinucleotide repeats. To better understand the multiple roles of the Srs2 helicase during these processes, we analysed the ability of Srs2 to bind and unwind various DNA substrates that mimic structures present during DNA replication and recombination. While leading or lagging strands were efficiently unwound, the presence of ssDNA binding protein RPA presented an obstacle for Srs2 translocation. We also tested the preferred directionality of unwinding of various substrates and studied the effect of Rad51 and Mre11 proteins on Srs2 helicase activity. These biochemical results help us understand the possible role of Srs2 in the processing of stalled or blocked replication forks as a part of post-replication repair as well as homologous recombination (HR).     

Mre11 and Exo1 contribute to the initiation and processivity of resection at meiotic double-strand breaks made independently of Spo11

During meiosis DNA double-strand breaks (DSBs) are induced and repaired by homologous recombination to create gene conversion and crossover products. Mostly these DSBs are made by Spo11, which covalently binds to the DSB ends. More rarely in Saccharomyces cerevisiae, other meiotic DSBs are formed by self-homing endonucleases such as VDE, which is site specific and does not covalently bind to the DSB ends. We have used experimentally located VDE-DSB sites to analyse an intermediate step in homologous recombination, resection of the single-strand ending 5' at the DSB site. Analysis of strains with different mutant alleles of MRE11 (mre11-58S and mre11-H125N) and deleted for EXO1 indicated that these two nucleases make significant contributions to repair of VDE-DSBs. Physical analysis of single-stranded repair intermediates indicates that efficient initiation and processivity of resection at VDE-DSBs require both Mre11 and Exo1, with loss of function for either protein causing severe delay in resection. We propose that these experiments model what happens at Spo11-DSBs after removal of the covalently bound protein, and that Mre11 and Exo1 are the major nucleases involved in creating resection tracts of widely varying lengths typical of meiotic recombination.