Sumoylation regulates the stability and nuclease activity of Saccharomyces cerevisiae Dna2

Dna2 is an essential nuclease-helicase that acts in several distinct DNA metabolic pathways including DNA replication and recombination. To balance these functions and prevent unscheduled DNA degradation, Dna2 activities must be regulated. Here we show that Saccharomyces cerevisiae Dna2 function is controlled by sumoylation. We map the sumoylation sites to the N-terminal regulatory domain of Dna2 and show that in vitro sumoylation of recombinant Dna2 impairs its nuclease but not helicase activity. In cells, the total levels of the non-sumoylatable Dna2 variant are elevated. However, non-sumoylatable Dna2 shows impaired nuclear localization and reduced recruitment to foci upon DNA damage. Non-sumoylatable Dna2 reduces the rate of DNA end resection, as well as impedes cell growth and cell cycle progression through S phase. Taken together, these findings show that in addition to Dna2 phosphorylation described previously, Dna2 sumoylation is required for the homeostasis of the Dna2 protein function to promote genome stability

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

Main steps in DNA double-strand break repair: An introduction to homologous recombination and related processes

DNA double-strand breaks arise accidentally upon exposure of DNA to radiation, chemicals or result from faulty DNA metabolic processes. DNA breaks can also be introduced in a programmed manner, such as during the maturation of the immune system, meiosis or cancer chemo- or radiotherapy. Cells have developed a variety of repair pathways, which are fine-tuned to the specific needs of a cell. Accordingly, vegetative cells employ mechanisms that restore the integrity of broken DNA with the highest efficiency at the lowest cost of mutagenesis. In contrast, meiotic cells or developing lymphocytes exploit DNA breakage to generate diversity. Here, we review the main pathways of eukaryotic DNA double-strand break repair with the focus on homologous recombination and its various sub-pathways. We highlight the differences between homologous recombination and end-joining mechanisms including nonhomologous end-joining and microhomology-mediated end-joining, and offer insights into how these pathways are regulated. Finally, we introduce non-canonical functions of the recombination proteins, in particular during DNA replication stress

Sumoylation regulates the stability and nuclease activity of Saccharomyces cerevisiae Dna2

Dna2 is an essential nuclease-helicase that acts in several distinct DNA metabolic pathways including DNA replication and recombination. To balance these functions and prevent unscheduled DNA degradation, Dna2 activities must be regulated. Here we show that Saccharomyces cerevisiae Dna2 function is controlled by sumoylation. We map the sumoylation sites to the N-terminal regulatory domain of Dna2 and show that in vitro sumoylation of recombinant Dna2 impairs its nuclease but not helicase activity. In cells, the total levels of the non-sumoylatable Dna2 variant are elevated. However, non-sumoylatable Dna2 shows impaired nuclear localization and reduced recruitment to foci upon DNA damage. Non-sumoylatable Dna2 reduces the rate of DNA end resection, as well as impedes cell growth and cell cycle progression through S phase. Taken together, these findings show that in addition to Dna2 phosphorylation described previously, Dna2 sumoylation is required for the homeostasis of the Dna2 protein function to promote genome stability

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

A meiotic XPF-ERCC1-like complex recognizes joint molecule recombination intermediates to promote crossover formation

Meiotic crossover formation requires the stabilization of early recombination intermediates by a set of proteins and occurs within the environment of the chromosome axis, a structure important for the regulation of meiotic recombination events. The molecular mechanisms underlying and connecting crossover recombination and axis localization are elusive. Here, we identified the ZZS (Zip2–Zip4–Spo16) complex, required for crossover formation, which carries two distinct activities: one provided by Zip4, which acts as hub through physical interactions with components of the chromosome axis and the crossover machinery, and the other carried by Zip2 and Spo16, which preferentially bind branched DNA molecules in vitro. We found that Zip2 and Spo16 share structural similarities to the structure-specific XPF–ERCC1 nuclease, although it lacks endonuclease activity. The XPF domain of Zip2 is required for crossover formation, suggesting that, together with Spo16, it has a noncatalytic DNA recognition function. Our results suggest that the ZZS complex shepherds recombination intermediates toward crossovers as a dynamic structural module that connects recombination events to the chromosome axis. The identification of the ZZS complex improves our understanding of the various activities required for crossover implementation and is likely applicable to other organisms, including mammals

Main steps in DNA double-strand break repair: an introduction to homologous recombination and related processes

DNA double-strand breaks arise accidentally upon exposure of DNA to radiation and chemicals or result from faulty DNA metabolic processes. DNA breaks can also be introduced in a programmed manner, such as during the maturation of the immune system, meiosis, or cancer chemo- or radiotherapy. Cells have developed a variety of repair pathways, which are fine-tuned to the specific needs of a cell. Accordingly, vegetative cells employ mechanisms that restore the integrity of broken DNA with the highest efficiency at the lowest cost of mutagenesis. In contrast, meiotic cells or developing lymphocytes exploit DNA breakage to generate diversity. Here, we review the main pathways of eukaryotic DNA double-strand break repair with the focus on homologous recombination and its various subpathways. We highlight the differences between homologous recombination and end-joining mechanisms including non-homologous end-joining and microhomology-mediated end-joining and offer insights into how these pathways are regulated. Finally, we introduce noncanonical functions of the recombination proteins, in particular during DNA replication stress.ISSN:0009-5915ISSN:1432-088

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

Strand annealing and motor driven activities of SMARCAL1 and ZRANB3 are stimulated by RAD51 and the paralog complex

SMARCAL1, ZRANB3 and HLTF are required for the remodeling of replication forks upon stress to promote genome stability. RAD51, along with the RAD51 paralog complex, were also found to have recombination-independent functions in fork reversal, yet the underlying mechanisms remained unclear. Using reconstituted reactions, we build upon previous data to show that SMARCAL1, ZRANB3 and HLTF have unequal biochemical capacities, explaining why they have non-redundant functions. SMARCAL1 uniquely anneals RPA-coated ssDNA, which depends on its direct interaction with RPA, but not on ATP. SMARCAL1, along with ZRANB3, but not HLTF efficiently employ ATPase driven translocase activity to rezip RPA-covered bubbled DNA, which was proposed to mimic elements of fork reversal. In contrast, ZRANB3 and HLTF but not SMARCAL1 are efficient in branch migration that occurs downstream in fork remodeling. We also show that low concentrations of RAD51 and the RAD51 paralog complex, RAD51B-RAD51C-RAD51D-XRCC2 (BCDX2), directly stimulate the motor-driven activities of SMARCAL1 and ZRANB3 but not HLTF, and the interplay is underpinned by physical interactions. Our data provide a possible mechanism explaining previous cellular experiments implicating RAD51 and BCDX2 in fork reversal.ISSN:1362-4962ISSN:0301-561