The Saccaromyces cerevisiae Mlh1-Mlh3 heterodimer is an endonuclease that preferentially binds to Holliday junctions

MutLγ, a heterodimer of the MutL homologues Mlh1 and Mlh3, plays a critical role during meiotic homologous recombination. The meiotic function of Mlh3 is fully dependent on the integrity of a putative nuclease motif DQHA(X)2E(X)4E, inferring that the anticipated nuclease activity of Mlh1-Mlh3 is involved in the processing of joint molecules to generate crossover recombination products. Although a vast body of genetic and cell biological data regarding Mlh1-Mlh3 is available, mechanistic insights into its function have been lacking due to the unavailability of the recombinant protein complex. Here we expressed the yeast Mlh1-Mlh3 heterodimer and purified it into near homogeneity. We show that recombinant MutLγ is a nuclease that nicks double-stranded DNA. We demonstrate that MutLγ binds DNA with a high affinity, and shows a marked preference for Holliday junctions. We also expressed the human MLH1-MLH3 complex and show that preferential binding to Holliday junctions is a conserved capacity of eukaryotic MutLγ complexes. Specific DNA recognition has never been observed with any other eukaryotic MutL homologue. MutLγ thus represents a new paradigm for the function of the eukaryotic MutL protein family. We provide insights into the mode of Holliday junction recognition, and show that Mlh1-Mlh3 prefers to bind the open unstacked Holliday junction form. This further supports the model where MutLγ is part of a complex acting on joint molecules to generate crossovers in meiosis

The Mre11-Nbs1 Interface Is Essential for Viability and Tumor Suppression

The Mre11 complex (Mre11, Rad50, and Nbs1) is integral to both DNA repair and ataxia telangiectasia mutated (ATM)-dependent DNA damage signaling. All three Mre11 complex components are essential for viability at the cellular and organismal levels. To delineate essential and non-essential Mre11 complex functions that are mediated by Nbs1, we used TALEN-based genome editing to derive Nbs1 mutant mice (Nbs1mid mice), which harbor mutations in the Mre11 interaction domain of Nbs1. Nbs1mid alleles that abolished interaction were incompatible with viability. Conversely, a 108-amino-acid Nbs1 fragment comprising the Mre11 interface was sufficient to rescue viability and ATM activation in cultured cells and support differentiation of hematopoietic cells in vivo. These data indicate that the essential role of Nbs1 is via its interaction with Mre11 and that most of the Nbs1 protein is dispensable for Mre11 complex functions and suggest that Mre11 and Rad50 directly activate ATM

The Mre11-Nbs1 interface is essential for viability and tumor suppression

The Mre11 complex (Mre11, Rad50, and Nbs1) is integral to both DNA repair and ataxia telangiectasia mutated (ATM)-dependent DNA damage signaling. All three Mre11 complex components are essential for viability at the cellular and organismal levels. To delineate essential and non-essential Mre11 complex functions that are mediated by Nbs1, we used TALEN-based genome editing to derive Nbs1 mutant mice (Nbs1mid mice), which harbor mutations in the Mre11 interaction domain of Nbs1. Nbs1mid alleles that abolished interaction were incompatible with viability. Conversely, a 108-amino-acid Nbs1 fragment comprising the Mre11 interface was sufficient to rescue viability and ATM activation in cultured cells and support differentiation of hematopoietic cells in vivo. These data indicate that the essential role of Nbs1 is via its interaction with Mre11 and that most of the Nbs1 protein is dispensable for Mre11 complex functions and suggest that Mre11 and Rad50 directly activate ATM

NBS1 promotes the endonuclease of the MRE11-RAD50 complex by sensing CtIP phosphorylation

DNA end resection initiates DNA break repair by homologous recombination. MRE11-RAD50-NBS1 and phosphorylated CtIP perform the first resection step by MRE11-catalyzed endonucleolytic DNA cleavage. Human NBS1, more than its Xrs2 homologue from Saccharomyces cerevisiae, is crucial for this process, highlighting complex mechanisms that regulate the MRE11 nuclease in high eukaryotes. Using a reconstituted system, we show here that NBS1, through its FHA and BRCT domains, functions as a sensor of CtIP phosphorylation. NBS1 then activates the MRE11-RAD50 nuclease through direct physical interactions with MRE11. In absence of NBS1, MRE11-RAD50 exhibits a weaker nuclease activity, which requires CtIP but not strictly its phosphorylation. This identifies at least two mechanisms by which CtIP promotes MRE11: a phosphorylation-dependent mode through NBS1, and a phosphorylation-independent mode without NBS1. In support, we show that limited DNA end resection in absence of the FHA and BRCT domains of NBS1 occurs in vivo. Collectively, our data suggest that NBS1 restricts the MRE11- RAD50 nuclease to S-G2 phase when CtIP is extensively phosphorylated. This defines mechanisms that regulate the MRE11 nuclease in DNA metabolism

Restoration of replication fork stability in BRCA1- and BRCA2-deficient cells by inactivation of SNF2-family fork remodelers

To ensure the completion of DNA replication and maintenance of genome integrity, DNA repair factors protect stalled replication forks upon replication stress. Previous studies have identified a critical role for the tumor suppressors BRCA1 and BRCA2 in preventing the degradation of nascent DNA by the MRE11 nuclease after replication stress. Here we show that depletion of SMARCAL1, a SNF2-family DNA translocase that remodels stalled forks, restores replication fork stability and reduces the formation of replication stress-induced DNA breaks and chromosomal aberrations in BRCA1/2-deficient cells. In addition to SMARCAL1, other SNF2-family fork remodelers, including ZRANB3 and HLTF, cause nascent DNA degradation and genomic instability in BRCA1/2-deficient cells upon replication stress. Our observations indicate that nascent DNA degradation in BRCA1/2-deficient cells occurs as a consequence of MRE11-dependent nucleolytic processing of reversed forks generated by fork remodelers. These studies provide mechanistic insights into the processes that cause genome instability in BRCA1/2- deficient cells

SAMHD1 promotes DNA end resection to facilitate DNA repair by homologous recombination

DNA double-strand break (DSB) repair by homologous recombination (HR) is initiated by CtIP/MRN-mediated DNA end resection to maintain genome integrity. SAMHD1 is a dNTP triphosphohydrolase, which restricts HIV- 1 infection, and mutations are associated with Aicardi-Goutières syndrome and cancer. We show that SAMHD1 has a dNTPase-independent function in promoting DNA end resection to facilitate DSB repair by HR. SAMHD1 deficiency or Vpx-mediated degradation causes hypersensitivity to DSB-inducing agents, and SAMHD1 is recruited to DSBs. SAMHD1 complexes with CtIP via a conserved C-terminal domain and recruits CtIP to DSBs to facilitate end resection and HR. Significantly, a cancer-associated mutant with impaired CtIP interaction, but not dNTPase-inactive SAMHD1, fails to rescue the end resection impairment of SAMHD1 depletion. Our findings define a dNTPase-independent function for SAMHD1 in HR-mediated DSB repair by facilitating CtIP accrual to promote DNA end resection, providing insight into how SAMHD1 promotes genome integrity

From resection to resolution: biochemical investigation of early and late steps of homologous recombination

Cells utilize two major pathways to repair DNA double strand breaks (DSB) including homologous recombination (HR) and non-homologous end joining (NHEJ). DNA end resection of the 5'-terminated DNA at DSBs is the key event, which mechanistically determines the repair pathway choice by promoting HR while inhibiting NHEJ. Numerous studies have established the role of the MRE11-RAD50-NBS1 (MRN) complex and CtIP in the initiation of resection. However, the nucleolytic polarity of the only nuclease in the complex, MRE11, is the opposite (3' to 5') to the direction of resection (5' to 3'). To solve this enigma, a so-called bidirectional resection model has been proposed. MRE11 was anticipated to make incision(s) close to the DSB by its endonucleolytic activity, which creates an entry site for its 3' to 5' exonuclease that proceeds back towards the DSB. Although this model solves the polarity paradox, the mechanistic understanding of such nicking activity by MRE11 was undefined. Here I show that CtIP stimulates the endonuclease activity of MRN on double-stranded DNA (dsDNA). Phosphorylation of CtIP (pCtIP) at T847 is absolutely required for this stimulatory effect. The blocking of DNA ends, especially at the 5' end, is required to observe this MRN-pCtIP activity. In agreement with the proposed model, the position of the first incision by MRN-pCtIP complex maps to [symbol about] 20 nucleotides away from the 5' DNA end. The nicking activity is intrinsic to MRE11, as nuclease-dead (H129L D130V) variant of MRE11 does not show any clipping activity. Additionally, RAD50 mutants deficient in ATP binding (RAD50K42A) or ATP hydrolysis (RAD50K42R), fail to show any clipping activity, indicating the essential role of RAD50 and its ATPase activity. Interestingly, the removal of NBS1 from MRN complex results in the loss of the clipping activity. Therefore, unlike in yeast, my work establishes NBS1 as the indispensable component of the MRN complex together with RAD50 to stimulate MRE11-catalyzed clipping activity in humans. CtIP tetramerizes by making a dimer of dimers through its amino-terminus and such oligomerization is important for its in vivo functions in HR. The analysis of pCtIP L27E mutant, in which tetramerization is abolished while dimerization is preserved, revealed a reduced capacity to promote MRN, suggesting that proper oligomeric structure of CtIP is likely essential for its optimal activity. Furthermore, the deletion of 160 amino acids from the amino-terminus of CtIP (1-160∆ pCtIP), which disrupts the CtIP dimerization and consequently its tetramerization as well, reduces the pCtIP capacity to stimulate MRN drastically. In contrast to yeast, pCtIP also stimulates the MRN endonuclease on ssDNA circular plasmid. In terms of cofactors, Mg2+, Mn2+ and ATP were found to be important for the optimum activity of of MRN-pCtIP. My second project was focused on meiotic homologous recombination. Meiotic HR favours the formation of double Holliday junctions (dHJ) as intermediates of programmed DSB repair. These are, by a yet unknown mechanism, processed exclusively to preferentially to crossovers (CO to facilitate the production of genetic diversity. Meiotic cells have a dedicated and biased mechanism in place to ensure the formation of obligate COs. MLH1-MLH3 (MutLγ) has been strongly implicated in meiosis as a putative endonuclease responsible for cleaving dHJs in a biased manner to produces COs. Additionally, MSH4-MSH5 (MutSγ) has also been shown to function in the MutLγ mediated pathway. Despite the availability of extensive genetic data, the mechanistic understanding of the biased cleavage of dHJs by MutLγ and its partners remains elusive. Here, in collaboration with Nicolas Weyland, I set out to study and characterize the biochemical behaviour of human MLH1-MLH3 in conjunction with human MSH4-MSH5. Previously, we showed that hMutLγ prefers binding to HJs and similar structures. Further analysis indicated that it presumably binds to the core of a HJ. Here I could show that hMutLγ nicks super-coiled dsDNA non-specifically in the presence of Mn2+. No such activity with nuclease-dead hMutLγ (MLH1-MLH3D1223N) was observed, proving that the observed activity is intrinsic to hMutLγ. The presence of ATP further simulates the nicking activity of hMutLγ. Using purified hMutSγ, I could confirm that it prefers binding to HJs over dsDNA and slides upon HJ arms upon ATP binding. We could also show that hMutLγ directly interacts with hMutSγ in vitro. Furthermore, DNA binding analysis of hMutLγ and hMutSγ revealed that hMutLγ binds cooperatively to HJ with hMutSγ. No such observation with dsDNA emphasizes the specific nature of the observed effect with HJ. The data also indicates that hMutSγ further stabilizes the hMutLγ-DNA complex

Methods to Study DNA End Resection II: Biochemical Reconstitution Assays

DNA end resection initiates the largely accurate repair of DNA double-strand breaks (DSBs) by homologous recombination. Specifically, recombination requires the formation of 3' overhangs at DSB sites, which is carried out by nucleases that specifically degrade 5'-terminated DNA. In most cases, DNA end resection is a two-step process, comprising of initial short-range followed by more processive long-range resection. In this chapter, we describe selected assays that reconstitute both the short- and long-range pathways. First, we define methods to study the exonuclease and endonuclease activities of the MRE11-RAD50-NBS1 (MRN) complex in conjunction with phosphorylated cofactor CtIP. This reaction is particularly important to initiate processing of DNA breaks and to recruit components belonging to the subsequent long-range pathway. Next, we describe assays that reconstitute the concerted reactions of Bloom (BLM) or Werner (WRN) helicases that function together with the DNA2 nuclease-helicase, and which are as a complex capable to resect DNA of kilobases in length. The reconstituted reactions allow us to understand how the resection pathways function at the molecular level. The assays will be invaluable to define regulatory mechanisms and to identify inhibitory compounds, which may be valuable in cancer therapy

Methods to Study DNA End Resection I: Recombinant Protein Purification

Accurate repair of DNA double-strand breaks (DSBs) is carried out by homologous recombination. In order to repair DNA breaks by the recombination pathway, the 5'-terminated DNA strand at DSB sites must be first nucleolytically processed to produce 3'-overhang. The process is termed DNA end resection and involves the interplay of several nuclease complexes. DNA end resection commits DSB repair to the recombination pathway including a process termed single-strand annealing, as resected DNA ends are generally nonligatable by the competing nonhomologous end-joining machinery. Biochemical reconstitution experiments provided invaluable mechanistic insights into the DNA end resection pathways. In this chapter, we describe preparation procedures of key proteins involved in DNA end resection in human cells, including the MRE11-RAD50-NBS1 complex, phosphorylated variant of CtIP, the DNA2 nuclease-helicase with its helicase partners Bloom (BLM) or Werner (WRN), as well as the single-stranded DNA-binding protein replication protein A. The availability of recombinant DNA end resection factors will help to further elucidate resection mechanisms and regulatory processes that may involve novel protein partners and posttranslational modifications

Phosphorylated CtIP functions as a co-factor of the MRE11-RAD50-NBS1 endonuclease in DNA end resection

To repair a DNA double-strand break (DSB) by homologous recombination (HR), the 5'-terminated strand of the DSB must be resected. The human MRE11-RAD50-NBS1 (MRN) and CtIP proteins were implicated in the initiation of DNA end resection, but the underlying mechanism remained undefined. Here, we show that CtIP is a co-factor of the MRE11 endonuclease activity within the MRN complex. This function is absolutely dependent on CtIP phosphorylation that includes the key cyclin-dependent kinase target motif at Thr-847. Unlike in yeast, where the Xrs2/NBS1 subunit is dispensable in vitro, NBS1 is absolutely required in the human system. The MRE11 endonuclease in conjunction with RAD50, NBS1, and phosphorylated CtIP preferentially cleaves 5'-terminated DNA strands near DSBs. Our results define the initial step of HR that is particularly relevant for the processing of DSBs bearing protein blocks