Mapping yeast mitotic 5′ resection at base resolution reveals the sequence and positional dependence of nucleases in vivo

Resection of the 5′-terminated strand at DNA double-strand breaks (DSBs) is the critical regulated step in the transition to homologous recombination. Recent studies have described a multi-step model of DSB resection where endonucleolytic cleavage mediated by Mre11 and Sae2 leads to further degradation mediated by redundant pathways catalyzed by Exo1 and Sgs1/Dna2. These models have not been well tested at mitotic DSBs in vivo because most methods used to monitor resection cannot precisely map early cleavage events. Here we report resection monitoring with high-throughput sequencing using molecular identifiers, allowing exact counting of cleaved 5′ ends at base resolution. Mutant strains, including exo1Δ, mre11-H125N and exo1Δ sgs1Δ, revealed a major Mre11-dependent cleavage position 60–70 bp from the DSB end whose exact position depended on local sequence. They further revealed an Exo1-dependent pause point approximately 200 bp from the DSB. Suppressing resection extension in exo1Δ sgs1Δ yeast exposed a footprint of regions where cleavage was restricted within 119 bp of the DSB. These results provide detailed in vivo views of prevailing models of DSB resection and extend them to show the combined influence of sequence specificity and access restrictions on Mre11 and Exo1 nucleases.     

Dna2 Is Involved in CA Strand Resection and Nascent Lagging Strand Completion at Native Yeast Telomeres

Post-replicational telomere end processing involves both extension by telomerase and resection to produce 3′-GT-overhangs that extend beyond the complementary 5′-CA-rich strand. Resection must be carefully controlled to maintain telomere length. At short de novo telomeres generated artificially by HO endonuclease in the G₂ phase, we show that dna2-defective strains are impaired in both telomere elongation and sequential 5′-CA resection. At native telomeres in dna2 mutants, GT-overhangs do clearly elongate during late S phase but are shorter than in wild type, suggesting a role for Dna2 in 5′-CA resection but also indicating significant redundancy with other nucleases. Surprisingly, elimination of Mre11 nuclease or Exo1, which are complementary to Dna2 in resection of internal double strand breaks, does not lead to further shortening of GT-overhangs in dna2 mutants. A second step in end processing involves filling in of the CA-strand to maintain appropriate telomere length. We show that Dna2 is required for normal telomeric CA-strand fill-in. Yeast dna2 mutants, like mutants in DNA ligase 1 (cdc9), accumulate low molecular weight, nascent lagging strand DNA replication intermediates at telomeres. Based on this and other results, we propose that FEN1 is not sufficient and that either Dna2 or Exo1 is required to supplement FEN1 in maturing lagging strands at telomeres. Telomeres may be among the subset of genomic locations where Dna2 helicase/nuclease is essential for the two-nuclease pathway of primer processing on lagging strands.     

Inviability of a DNA2 deletion mutant is due to the DNA damage checkpoint

Dna2 is a dual polarity exo/endonuclease, and 5′ to 3′ DNA helicase involved in Okazaki Fragment Processing (OFP) and Double-Strand Break (DSB) Repair. In yeast, DNA2 is an essential gene, as expected for a DNA replication protein. Suppression of the lethality of dna2Δ mutants has been found to occur by two mechanisms: overexpression of RAD27^scFEN1, encoding a 5′ to 3′ exo/endo nuclease that processes Okazaki fragments (OFs) for ligation, or deletion of PIF1, a 5′ to 3′ helicase involved in mitochondrial recombination, telomerase inhibition and OFP. Mapping of a novel, spontaneously arising suppressor of dna2Δ now reveals that mutation of rad9 and double mutation of rad9 mrc1 can also suppress the lethality of dna2Δ mutants. Interaction of dna2Δ and DNA damage checkpoint mutations provides insight as to why dna2Δ is lethal but rad27Δ is not, even though evidence shows that Rad27^ScFEN1 processes most of the Okazaki fragments, while Dna2 processes only a subset.Key words: yeast, RAD27, RAD9, RAD53, Okazaki fragment processing, DNA replication, exo1     

Functional Interplay between the 53BP1-Ortholog Rad9 and the Mre11 Complex Regulates Resection,End-Tethering and Repair of a Double-Strand Break

The Mre11-Rad50-Xrs2 nuclease complex, together with Sae2, initiates the 5′-to-3′ resection of Double-Strand DNA Breaks (DSBs). Extended 3′ single stranded DNA filaments can be exposed from a DSB through the redundant activities of the Exo1 nuclease and the Dna2 nuclease with the Sgs1 helicase. In the absence of Sae2, Mre11 binding to a DSB is prolonged, the two DNA ends cannot be kept tethered, and the DSB is not efficiently repaired. Here we show that deletion of the yeast 53BP1-ortholog RAD9 reduces Mre11 binding to a DSB, leading to Rad52 recruitment and efficient DSB end-tethering, through an Sgs1-dependent mechanism. As a consequence, deletion of RAD9 restores DSB repair either in absence of Sae2 or in presence of a nuclease defective MRX complex. We propose that, in cells lacking Sae2, Rad9/53BP1 contributes to keep Mre11 bound to a persistent DSB, protecting it from extensive DNA end resection, which may lead to potentially deleterious DNA deletions and genome rearrangements.     

The pattern of sensitivity of yeast dna2 mutants to DNA damaging agents suggests a role in DSB and postreplication repair pathways

The Saccharomyces cerevisiae DNA2 gene encodes a DNA-stimulated ATPase and DNA helicase/nuclease essential for DNA replication. In characterizing dna2 mutants, we have found that Dna2p also participates in DNA repair or in damage avoidance mechanisms. dna2 mutants are sensitive to X rays, although they are less sensitive than rad52 mutants. The X-ray sensitivity of dna2 mutants is suppressed by overexpression of a 5' to 3' exonuclease, the yeast FEN-1 structure-specific nuclease, encoded by the RAD27 gene, which also suppresses the growth defect of dna2-ts mutants. SGS1 encodes a helicase with similar properties to Dna2 protein. Although sgs1Delta mutants are resistant to X rays, dna2-2 sgs1Delta double mutants are more sensitive to X rays than the dna2-2 mutant. Temperature sensitive dna2 mutants are only slightly sensitive to UV light, show normal levels of spontaneous and UV induced mutagenesis, and have only a 2.5-fold elevated level of dinucleotide tract instability compared to wildtype. However, dna2Delta strains kept alive by overproduction of RAD27 are highly sensitive to UV light. These phenotypes, in addition to the epistasis analysis reported, allow us to propose that Dna2 is involved in postreplication and DSB repair pathways.     

NurA,a novel 5'-3' nuclease gene linked to rad50 and mre11 homologs of thermophilic Archaea

We isolated and characterized a new nuclease (NurA) exhibiting both single-stranded endonuclease activity and 5′–3′ exonuclease activity on single-stranded and double-stranded DNA from the hyperthermophilic archaeon Sulfolobus acidocaldarius. Nuclease homologs are detected in all thermophilic archaea and, in most species, the nurA gene is organized in an operon-like structure with rad50 and mre11 archaeal homologs. This nuclease might thus act in concert with Rad50 and Mre11 proteins in archaeal recombination/repair. To our knowledge, this is the first report of a 5′–3′ nuclease potentially associated with Rad50 and Mre11-like proteins that may lead to the processing of double-stranded breaks in 3′ single-stranded tails.     

Alteration of gene conversion tract length and associated crossing over during plasmid gap repair in nuclease-deficient strains of Saccharomyces cerevisiae

A plasmid gap repair assay was used to assess the role of three known nucleases, Exo1, Mre11 and Rad1, in the processing of DNA ends and resolution of recombination intermediates during double-strand gap repair. In this assay, alterations in end processing or branch migration are reflected by the frequency of co-conversion of a chromosomal marker 200 bp from the gap. Gap repair associated with crossing over results in integration at the homologous chromosomal locus, whereas the plasmid remains episomal for non-crossover repair events. In mre11 strains, the frequency of gap repair was reduced 3- to 10-fold and conversion tracts were shorter than in the wild-type strain, consistent with a role for this nuclease in processing double-strand breaks. However, conversion tracts were longer in a strain containing the nuclease deficient allele, mre11-H125N, suggesting increased end processing by redundant nucleases. The frequency of gap repair was reduced 2-fold in rad1 mutants and crossing over was reduced, consistent with a role for Rad1 in cleaving recombination intermediates. The frequency of gap repair was increased in exo1 mutants with a significant increase in crossing over. In exo1 mre11 double mutants gap repair was reduced to below the mre11 single mutant level.     

Human Dna2 Is a Nuclear and Mitochondrial DNA Maintenance Protein

Dna2 is a highly conserved helicase/nuclease that in yeast participates in Okazaki fragment processing, DNA repair, and telomere maintenance. Here, we investigated the biological function of human Dna2 (hDna2). Immunofluorescence and biochemical fractionation studies demonstrated that hDna2 was present in both the nucleus and the mitochondria. Analysis of mitochondrial hDna2 revealed that it colocalized with a subfraction of DNA-containing mitochondrial nucleoids in unperturbed cells. Upon the expression of disease-associated mutant forms of the mitochondrial Twinkle helicase which induce DNA replication pausing/stalling, hDna2 accumulated within nucleoids. RNA interference-mediated depletion of hDna2 led to a modest decrease in mitochondrial DNA replication intermediates and inefficient repair of damaged mitochondrial DNA. Importantly, hDna2 depletion also resulted in the appearance of aneuploid cells and the formation of internuclear chromatin bridges, indicating that nuclear hDna2 plays a role in genomic DNA stability. Together, our data indicate that hDna2 is similar to its yeast counterpart and is a new addition to the growing list of proteins that participate in both nuclear and mitochondrial DNA maintenance.DNA damage arises from errors in the replication process, as well as a myriad of intrinsic and extrinsic DNA-damaging agents that continually assault cells. Failure to efficiently repair DNA lesions leads to accumulation of mutations that contribute to numerous pathologies, including carcinogenesis. In addition to genomic DNA, mitochondrial DNA (mtDNA) is subject to damage that requires repair to maintain integrity. For these reasons, it is not surprising that DNA replication and repair proteins display significant plasticity that allows participation in several divergent replication and repair processes. In addition, numerous mechanisms, including alternative splicing, posttranslational modifications, or utilization of alternative translation initiation start sites, allow DNA replication and repair proteins such as Pif1, DNA ligase III, and APE1 to localize to the nucleus and the mitochondrion and participate in DNA replication and/or repair (9, 17, 25), thus ensuring genomic DNA and mtDNA integrity.Dna2 is an evolutionarily conserved helicase/nuclease enzyme. Originally discovered in Saccharomyces cerevisiae, Dna2 orthologs are found throughout the animal kingdom, including humans (5, 22, 28). Early studies demonstrated that Dna2 functions in concert with Flap endonuclease 1 (FEN1) to remove long DNA flaps that form upon lagging-strand DNA replication (6). However, in contrast to FEN1, Dna2 is an essential gene in yeast, suggesting that other proteins, including FEN1, cannot compensate for its loss in DNA replication or that it possesses functions beyond its role in Okazaki fragment processing. In agreement with this, genetic and biochemical studies have implicated Dna2 in DNA double-strand break (DSB) repair, telomere regulation, and mitochondrial function (8, 10, 15, 26, 38, 44, 45).Analysis of Dna2 in yeast revealed that it undergoes dynamic cell cycle localization. Dna2 localizes to telomeres during G₁, relocalizes throughout the genome in S phase, and moves back to the telomere during late S/G₂, where it participates in telomere replication and telomerase-dependent telomere elongation (10). Dna2 also leaves the telomere following treatment with bleomycin and localizes to sites of DNA DSBs (10). In addition, dna2 mutants are sensitive to DNA damage induced by gamma radiation and methanesulfonic acid methyl ester (7, 15). These phenotypes may be explained by recent work demonstrating that Dna2 plays an important role in 5′-end resection following DSBs. Indeed, upon induction of DSBs and initiation of 5′-end resection by the Mre11-Rad50-Xrs2 complex, Dna2 and Sgs1 cooperate to further degrade the 5′ end, creating long 3′ strands essential for homologous recombination (26, 45). Finally, while dna2Δ mutations are lethal in budding yeast, the dna2Δ pif1-m2 (nuclear PIF1) double mutations rescue dna2Δ lethality but produce a petite phenotype, suggesting that Dna2 is also involved in mtDNA maintenance (8).Recently, the human ortholog of Dna2 was cloned and characterized (23, 29). Biochemical analysis revealed that, similar to its yeast counterpart, the human Dna2 (hDna2) protein possesses nuclease, ATPase, and limited helicase activities (23, 29), suggesting that it carries out analogous functions in yeast and mammalian cells. However, hDna2''s putative role in genomic DNA repair and replication was called into question by a recent study suggesting that hDna2 is absent from the nucleus and found exclusively within the mitochondria, where it participates in mtDNA repair (44). Further in vitro biochemical studies suggested that hDna2 also participates in mtDNA replication (44). Here, we confirm that hDna2 localizes to the mitochondria and demonstrate that hDna2 participates in mtDNA replication and repair. However, our studies go further by uncovering a nuclear form of hDna2 that plays an important role in genomic stability. Indeed, we demonstrate that depletion of hDna2 leads to the appearance of aneuploid cells and the formation of internuclear chromatin bridges, indicating that hDna2, like its yeast counterpart, is essential to maintain nuclear DNA stability.     

DNA2 Cooperates with the WRN and BLM RecQ Helicases to Mediate Long-range DNA End Resection in Human Cells

The 5′-3′ resection of DNA ends is a prerequisite for the repair of DNA double strand breaks by homologous recombination, microhomology-mediated end joining, and single strand annealing. Recent studies in yeast have shown that, following initial DNA end processing by the Mre11-Rad50-Xrs2 complex and Sae2, the extension of resection tracts is mediated either by exonuclease 1 or by combined activities of the RecQ family DNA helicase Sgs1 and the helicase/endonuclease Dna2. Although human DNA2 has been shown to cooperate with the BLM helicase to catalyze the resection of DNA ends, it remains a matter of debate whether another human RecQ helicase, WRN, can substitute for BLM in DNA2-catalyzed resection. Here we present evidence that WRN and BLM act epistatically with DNA2 to promote the long-range resection of double strand break ends in human cells. Our biochemical experiments show that WRN and DNA2 interact physically and coordinate their enzymatic activities to mediate 5′-3′ DNA end resection in a reaction dependent on RPA. In addition, we present in vitro and in vivo data suggesting that BLM promotes DNA end resection as part of the BLM-TOPOIIIα-RMI1-RMI2 complex. Our study provides new mechanistic insights into the process of DNA end resection in mammalian cells.     

The Nuclease Activity of Mre11 Is Required for Meiosis but Not for Mating Type Switching, End Joining, or Telomere Maintenance

The Saccharomyces cerevisiae MRE11 gene is required for the repair of ionizing radiation-induced DNA damage and for the initiation of meiotic recombination. Sequence analysis has revealed homology between Mre11 and SbcD, the catalytic subunit of an Escherichia coli enzyme with endo- and exonuclease activity, SbcCD. In this study, the purified Mre11 protein was found to have single-stranded endonuclease activity. This activity was absent from mutant proteins containing single amino acid substitutions in either one of two sequence motifs that are shared by Mre11 and SbcD. Mutants with allele mre11-D56N or mre11-H125N were partially sensitive to ionizing radiation but lacked the other mitotic phenotypes of poor vegetative growth, hyperrecombination, defective nonhomologous end joining, and shortened telomeres that are characteristic of the mre11 null mutant. Diploids homozygous for the mre11-H125N mutation failed to sporulate and accumulated unresected double-strand breaks (DSB) during meiosis. We propose that in mitotic cells DSBs can be processed by other nucleases that are partially redundant with Mre11, but these activities are unable to process Spo11-bound DSBs in meiotic cells.     

Structural and functional analysis of Mre11-3

The Mre11, Rad50 and Nbs1 proteins make up the conserved multi-functional Mre11 (MRN) complex involved in multiple, critical DNA metabolic processes including double-strand break repair and telomere maintenance. The Mre11 protein is a nuclease with broad substrate recognition, but MRN-dependent processes requiring the nuclease activity are not clearly defined. Here, we report the functional and structural characterization of a nuclease-deficient Mre11 protein termed mre11-3. Importantly, the hmre11-3 protein has wild-type ability to bind DNA, Rad50 and Nbs1; however, nuclease activity was completely abrogated. When expressed in cell lines from patients with ataxia telangiectasia-like disorder (ATLD), hmre11-3 restored the formation of ionizing radiation-induced foci. Consistent with the biochemical results, the 2.3 Å crystal structure of mre11-3 from Pyrococcus furiosus revealed an active site structure with a wild-type-like metal-binding environment. The structural analysis of the H85L mutation provides a detailed molecular basis for the ability of mre11-3 to bind but not hydrolyze DNA. Together, these results establish that the mre11-3 protein provides an excellent system for dissecting nuclease-dependent and independent functions of the Mre11 complex.     

S. cerevisiae Mre11 recruits conjugated SUMO moieties to facilitate the assembly and function of the Mre11-Rad50-Xrs2 complex

Double-strand breaks (DSBs) in chromosomes are the most challenging type of DNA damage. The yeast and mammalian Mre11-Rad50-Xrs2/Nbs1 (MRX/N)-Sae2/Ctp1 complex catalyzes the resection of DSBs induced by secondary structures, chemical adducts or covalently-attached proteins. MRX/N also initiates two parallel DNA damage responses—checkpoint phosphorylation and global SUMOylation—to boost a cell''s ability to repair DSBs. However, the molecular mechanism of this SUMO-mediated response is not completely known. In this study, we report that Saccharomyces cerevisiae Mre11 can non-covalently recruit the conjugated SUMO moieties, particularly the poly-SUMO chain. Mre11 has two evolutionarily-conserved SUMO-interacting motifs, Mre11^SIM1 and Mre11^SIM2, which reside on the outermost surface of Mre11. Mre11^SIM1 is indispensable for MRX assembly. Mre11^SIM2 non-covalently links MRX with the SUMO enzymes (E2/Ubc9 and E3/Siz2) to promote global SUMOylation of DNA repair proteins. Mre11^SIM2 acts independently of checkpoint phosphorylation. During meiosis, the mre11^SIM2 mutant, as for mre11S, rad50S and sae2Δ, allows initiation but not processing of Spo11-induced DSBs. Using MRX and DSB repair as a model, our work reveals a general principle in which the conjugated SUMO moieties non-covalently facilitate the assembly and functions of multi-subunit protein complexes.     

Role of the nuclease activity of Saccharomyces cerevisiae Mre11 in repair of DNA double-strand breaks in mitotic cells

The Rad50:Mre11:Xrs2 (RMX) complex functions in repair of DNA double-strand breaks (DSBs) by recombination and nonhomologous end-joining (NHEJ) and is also required for telomere stability. The Mre11 subunit exhibits nuclease activities in vitro, but the role of these activities in repair in mitotic cells has not been established. In this study we have performed a comparative study of three mutants (mre11-D16A, -D56N, and -H125N) previously shown to have reduced nuclease activities in vitro. In ends-in and ends-out chromosome recombination assays using defined plasmid and oligonucleotide DNA substrates, mre11-D16A cells were as deficient as mre11 null strains, but defects were small in mre11-D56N and -H125N mutants. mre11-D16A cells, but not the other mutants, also displayed strong sensitivity to ionizing radiation, with residual resistance largely dependent on the presence of the partially redundant nuclease Exo1. mre11-D16A mutants were also most sensitive to the S-phase-dependent clastogens hydroxyurea and methyl methanesulfonate but, as previously observed for D56N and H125N mutants, were not defective in NHEJ. Importantly, the affinity of purified Mre11-D16A protein for Rad50 and Xrs2 was indistinguishable from wild type and the mutant protein formed complexes with equivalent stoichiometry. Although the role of the nuclease activity has been questioned in previous studies, the comparative data presented here suggest that the nuclease function of Mre11 is required for RMX-mediated recombinational repair and telomere stabilization in mitotic cells.     

The Role of MRN in the S-Phase DNA Damage Checkpoint Is Independent of Its Ctp1-dependent Roles in Double-Strand Break Repair and Checkpoint Signaling

The Mre11-Rad50-Nbs1 (MRN) complex has many biological functions: processing of double-strand breaks in meiosis, homologous recombination, telomere maintenance, S-phase checkpoint, and genome stability during replication. In the S-phase DNA damage checkpoint, MRN acts both in activation of checkpoint signaling and downstream of the checkpoint kinases to slow DNA replication. Mechanistically, MRN, along with its cofactor Ctp1, is involved in 5′ resection to create single-stranded DNA that is required for both signaling and homologous recombination. However, it is unclear whether resection is essential for all of the cellular functions of MRN. To dissect the various roles of MRN, we performed a structure–function analysis of nuclease dead alleles and potential separation-of-function alleles analogous to those found in the human disease ataxia telangiectasia-like disorder, which is caused by mutations in Mre11. We find that several alleles of rad32 (the fission yeast homologue of mre11), along with ctp1Δ, are defective in double-strand break repair and most other functions of the complex, but they maintain an intact S phase DNA damage checkpoint. Thus, the MRN S-phase checkpoint role is separate from its Ctp1- and resection-dependent role in double-strand break repair. This observation leads us to conclude that other functions of MRN, possibly its role in replication fork metabolism, are required for S-phase DNA damage checkpoint function.     

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.     

Evidence suggesting that Pif1 helicase functions in DNA replication with the Dna2 helicase/nuclease and DNA polymerase delta

The precise machineries required for two aspects of eukaryotic DNA replication, Okazaki fragment processing (OFP) and telomere maintenance, are poorly understood. In this work, we present evidence that Saccharomyces cerevisiae Pif1 helicase plays a wider role in DNA replication than previously appreciated and that it likely functions in conjunction with Dna2 helicase/nuclease as a component of the OFP machinery. In addition, we show that Dna2, which is known to associate with telomeres in a cell-cycle-specific manner, may be a new component of the telomere replication apparatus. Specifically, we show that deletion of PIF1 suppresses the lethality of a DNA2-null mutant. The pif1delta dna2delta strain remains methylmethane sulfonate sensitive and temperature sensitive; however, these phenotypes can be suppressed by further deletion of a subunit of pol delta, POL32. Deletion of PIF1 also suppresses the cold-sensitive lethality and hydroxyurea sensitivity of the pol32delta strain. Dna2 is thought to function by cleaving long flaps that arise during OFP due to excessive strand displacement by pol delta and/or by an as yet unidentified helicase. Thus, suppression of dna2delta can be rationalized if deletion of POL32 and/or PIF1 results in a reduction in long flaps that require Dna2 for processing. We further show that deletion of DNA2 suppresses the long-telomere phenotype and the high rate of formation of gross chromosomal rearrangements in pif1Delta mutants, suggesting a role for Dna2 in telomere elongation in the absence of Pif1.     

BRCA1 and CtIP Are Both Required to Recruit Dna2 at Double-Strand Breaks in Homologous Recombination

Homologous recombination plays a key role in the repair of double-strand breaks (DSBs), and thereby significantly contributes to cellular tolerance to radiotherapy and some chemotherapy. DSB repair by homologous recombination is initiated by 5’ to 3’ strand resection (DSB resection), with nucleases generating the 3’ single-strand DNA (3’ssDNA) at DSB sites. Genetic studies of Saccharomyces cerevisiae demonstrate a two-step DSB resection, wherein CtIP and Mre11 nucleases carry out short-range DSB resection followed by long-range DSB resection done by Dna2 and Exo1 nucleases. Recent studies indicate that CtIP contributes to DSB resection through its non-catalytic role but not as a nuclease. However, it remains elusive how CtIP contributes to DSB resection. To explore the non-catalytic role, we examined the dynamics of Dna2 by developing an immuno-cytochemical method to detect ionizing-radiation (IR)-induced Dna2-subnuclear-focus formation at DSB sites in chicken DT40 and human cell lines. Ionizing-radiation induced Dna2 foci only in wild-type cells, but not in Dna2 depleted cells, with the number of foci reaching its maximum at 30 minutes and being hardly detectable at 120 minutes after IR. Induced foci were detectable in cells in the G2 phase but not in the G1 phase. These observations suggest that Dna2 foci represent the recruitment of Dna2 to DSB sites for DSB resection. Importantly, the depletion of CtIP inhibited the recruitment of Dna2 to DSB sites in both human cells and chicken DT40 cells. Likewise, a defect in breast cancer 1 (BRCA1), which physically interacts with CtIP and contributes to DSB resection, also inhibited the recruitment of Dna2. Moreover, CtIP physically associates with Dna2, and the association is enhanced by IR. We conclude that BRCA1 and CtIP contribute to DSB resection by recruiting Dna2 to damage sites, thus ensuring the robust DSB resection necessary for efficient homologous recombination.     

Biochemical analysis of human Dna2

Yeast Dna2 helicase/nuclease is essential for DNA replication and assists FEN1 nuclease in processing a subset of Okazaki fragments that have long single-stranded 5′ flaps. It is also involved in the maintenance of telomeres. DNA2 is a gene conserved in eukaryotes, and a putative human ortholog of yeast DNA2 (ScDNA2) has been identified. Little is known about the role of human DNA2 (hDNA2), although complementation experiments have shown that it can function in yeast to replace ScDNA2. We have now characterized the biochemical properties of hDna2. Recombinant hDna2 has single-stranded DNA-dependent ATPase and DNA helicase activity. It also has 5′–3′ nuclease activity with preference for single-stranded 5′ flaps adjacent to a duplex DNA region. The nuclease activity is stimulated by RPA and suppressed by steric hindrance at the 5′ end. Moreover, hDna2 shows strong 3′–5′ nuclease activity. This activity cleaves single-stranded DNA in a fork structure and, like the 5′–3′ activity, is suppressed by steric hindrance at the 3′-end, suggesting that the 3′–5′ nuclease requires a 3′ single-stranded end for activation. These biochemical specificities are very similar to those of the ScDna2 protein, but suggest that the 3′–5′ nuclease activity may be more important than previously thought.     

Role of DNA mismatch repair and double-strand break repair in genome stability and antifungal drug resistance in Candida albicans

Drug resistance has become a major problem in the treatment of Candida albicans infections. Genome changes, such as aneuploidy, translocations, loss of heterozygosity, or point mutations, are often observed in clinical isolates that have become resistant to antifungal drugs. To determine whether these types of alterations result when DNA repair pathways are eliminated, we constructed yeast strains bearing deletions in six genes involved in mismatch repair (MSH2 and PMS1) or double-strand break repair (MRE11, RAD50, RAD52, and YKU80). We show that the mre11Δ/mre11Δ, rad50Δ/rad50Δ, and rad52Δ/rad52Δ mutants are slow growing and exhibit a wrinkly colony phenotype and that cultures of these mutants contain abundant elongated pseudohypha-like cells. These same mutants are susceptible to hydrogen peroxide, tetrabutyl hydrogen peroxide, UV radiation, camptothecin, ethylmethane sulfonate, and methylmethane sulfonate. The msh2Δ/msh2Δ, pms1Δ/pms1Δ, and yku80Δ/yku80Δ mutants exhibit none of these phenotypes. We observed an increase in genome instability in mre11Δ/mre11Δ and rad50Δ/rad50Δ mutants by using a GAL1/URA3 marker system to monitor the integrity of chromosome 1. We investigated the acquisition of drug resistance in the DNA repair mutants and found that deletion of mre11Δ/mre11Δ, rad50Δ/rad50Δ, or rad52Δ/rad52Δ leads to an increased susceptibility to fluconazole. Interestingly, we also observed an elevated frequency of appearance of drug-resistant colonies for both msh2Δ/msh2Δ and pms1Δ/pms1Δ (MMR mutants) and rad50Δ/rad50Δ (DSBR mutant). Our data demonstrate that defects in double-strand break repair lead to an increase in genome instability, while drug resistance arises more rapidly in C. albicans strains lacking mismatch repair proteins or proteins central to double-strand break repair.