The absence of Top3 reveals an interaction between the Sgs1 and Pif1 DNA helicases in Saccharomyces cerevisiae

RecQ DNA helicases and Topo III topoisomerases have conserved genetic, physical, and functional interactions that are consistent with a model in which RecQ creates a recombination-dependent substrate that is resolved by Topo III. The phenotype associated with Topo III loss suggests that accumulation of a RecQ-created substrate is detrimental. In yeast, mutation of the TOP3 gene encoding Topo III causes pleiotropic defects that are suppressed by deletion of the RecQ homolog Sgs1. We searched for gene dosage suppressors of top3 and identified Pif1, a DNA helicase that acts with polarity opposite to that of Sgs1. Pif1 overexpression suppresses multiple top3 defects, but exacerbates sgs1 and sgs1 top3 defects. Furthermore, Pif1 helicase activity is essential in the absence of Top3 in an Sgs1-dependent manner. These data clearly demonstrate that Pif1 helicase activity is required to counteract Sgs1 helicase activity that has become uncoupled from Top3. Pif1 genetic interactions with the Sgs1-Top3 pathway are dependent upon homologous recombination. We also find that Pif1 is recruited to DNA repair foci and that the frequency of these foci is significantly increased in top3 mutants. Our results support a model in which Pif1 has a direct role in the prevention or repair of Sgs1-induced DNA damage that accumulates in top3 mutants.     

Sgs1--the maestro of recombination

The Sgs1 DNA helicase and its mammalian homolog BLM control crossover formation in mitotic cells. Zakharyevich et?al. and De Muyt et?al. now uncover a key role for Sgs1 in meiotic crossover regulation, which in turn reveals a joint molecule resolution pathway that produces the majority of crossovers in budding yeast.     

The genetic consequences of ablating helicase activity and the Top3 interaction domain of Sgs1

Sgs1, the RecQ helicase homolog, and Top3, the type-IA topoisomerase, physically interact and are required for genomic stability in budding yeast. Similarly, topoisomerase III genes physically pair with homologs of SGS1 in humans that are involved in the cancer predisposition and premature aging diseases Bloom, Werner, and Rothmund-Thompson syndromes. In the absence of Top1 activity, sgs1 mutants are severely growth impaired. Here, we investigate the role of Sgs1 helicase activity and its N-terminal Top3 interaction domain by using an allele-replacement technique to integrate mutant alleles at the native SGS1 genomic locus. We compare the phenotype of helicase-defective (sgs1-hd) and N-terminal deletion (sgs1-NDelta) strains to wild-type and sgs1 null strains. Like the sgs1 null, sgs1-hd mutations suppress top3 slow growth, cause a growth defect in the absence of Srs2 helicase, and impair meiosis. However, for recombination and the synthetic interaction with top1Delta mutations, loss of helicase activity exhibits a less severe phenotype than the null. Interestingly, deletion of the Top3 interaction domain of Sgs1 causes a top3-like phenotype, and furthermore, this effect is dependent on helicase activity. These results suggest that the protein-protein interaction between these two DNA-metabolism enzymes, even in the absence of helicase activity, is important for their function in catalyzing specific changes in DNA topology.     

Sgs1 function in the repair of DNA replication intermediates is separable from its role in homologous recombinational repair

Mutations in human homologues of the bacterial RecQ helicase cause diseases leading to cancer predisposition and/or shortened lifespan (Werner, Bloom, and Rothmund–Thomson syndromes). The budding yeast Saccharomyces cerevisiae has one RecQ helicase, Sgs1, which functions with Top3 and Rmi1 in DNA repair. Here, we report separation‐of‐function alleles of SGS1 that suppress the slow growth of top3Δ and rmi1Δ cells similar to an SGS1 deletion, but are resistant to DNA damage similar to wild‐type SGS1. In one allele, the second acidic region is deleted, and in the other, only a single aspartic acid residue 664 is deleted. sgs1‐D664Δ, unlike sgs1Δ, neither disrupts DNA recombination nor has synthetic growth defects when combined with DNA repair mutants. However, during S phase, it accumulates replication‐associated X‐shaped structures at damaged replication forks. Furthermore, fluorescent microscopy reveals that the sgs1‐D664Δ allele exhibits increased spontaneous RPA foci, suggesting that the persistent X‐structures may contain single‐stranded DNA. Taken together, these results suggest that the Sgs1 function in repair of DNA replication intermediates can be uncoupled from its role in homologous recombinational repair.     

Processing of homologous recombination repair intermediates by the Sgs1-Top3-Rmi1 and Mus81-Mms4 complexes

Homologous recombination repair (HRR) is an evolutionarily conserved cellular process that is important for the maintenance of genome stability during S phase. Inactivation of the Saccharomyces cerevisiae Sgs1-Top3-Rmi1 complex leads to the accumulation of unprocessed, X-shaped HRR intermediates (X structures) following replicative stress. Further characterization of these X structures may reveal why loss of BLM (the human Sgs1 ortholog) leads to the human cancer predisposition disorder, Bloom syndrome. In two recent complementary studies, we examined the nature of the X structures arising in yeast strains lacking Sgs1, Top3 or Rmi1 by identifying which proteins could process these structures in vivo. We revealed that the unprocessed X structures that accumulate in these strains could be resolved by the ectopic overexpression of two different Holliday junction (HJ) resolvases, and that the endogenous Mus81-Mms4 endonuclease could also remove them, albeit slowly. In this review, we discuss the implications of these results and review the putative roles for the Sgs1-Top3-Rmi1 and Mus81-Mms4 complexes in the processing of various types of HRR intermediates during S phase.     

Top2 and Sgs1-Top3 Act Redundantly to Ensure rDNA Replication Termination

Faithful DNA replication with correct termination is essential for genome stability and transmission of genetic information. Here we have investigated the potential roles of Topoisomerase II (Top2) and the RecQ helicase Sgs1 during late stages of replication. We find that cells lacking Top2 and Sgs1 (or Top3) display two different characteristics during late S/G2 phase, checkpoint activation and accumulation of asymmetric X-structures, which are both independent of homologous recombination. Our data demonstrate that checkpoint activation is caused by a DNA structure formed at the strongest rDNA replication fork barrier (RFB) during replication termination, and consistently, checkpoint activation is dependent on the RFB binding protein, Fob1. In contrast, asymmetric X-structures are formed independent of Fob1 at less strong rDNA replication fork barriers. However, both checkpoint activation and formation of asymmetric X-structures are sensitive to conditions, which facilitate fork merging and progression of replication forks through replication fork barriers. Our data are consistent with a redundant role of Top2 and Sgs1 together with Top3 (Sgs1-Top3) in replication fork merging at rDNA barriers. At RFB either Top2 or Sgs1-Top3 is essential to prevent formation of a checkpoint activating DNA structure during termination, but at less strong rDNA barriers absence of the enzymes merely delays replication fork merging, causing an accumulation of asymmetric termination structures, which are solved over time.     

Functional and physical interaction between Bcl-X(L) and a BH3-like domain in Beclin-1

The anti-apoptotic proteins Bcl-2 and Bcl-X(L) bind and inhibit Beclin-1, an essential mediator of autophagy. Here, we demonstrate that this interaction involves a BH3 domain within Beclin-1 (residues 114-123). The physical interaction between Beclin-1 and Bcl-X(L) is lost when the BH3 domain of Beclin-1 or the BH3 receptor domain of Bcl-X(L) is mutated. Mutation of the BH3 domain of Beclin-1 or of the BH3 receptor domain of Bcl-X(L) abolishes the Bcl-X(L)-mediated inhibition of autophagy triggered by Beclin-1. The pharmacological BH3 mimetic ABT737 competitively inhibits the interaction between Beclin-1 and Bcl-2/Bcl-X(L), antagonizes autophagy inhibition by Bcl-2/Bcl-X(L) and hence stimulates autophagy. Knockout or knockdown of the BH3-only protein Bad reduces starvation-induced autophagy, whereas Bad overexpression induces autophagy in human cells. Gain-of-function mutation of the sole BH3-only protein from Caenorhabditis elegans, EGL-1, induces autophagy, while deletion of EGL-1 compromises starvation-induced autophagy. These results reveal a novel autophagy-stimulatory function of BH3-only proteins beyond their established role as apoptosis inducers. BH3-only proteins and pharmacological BH3 mimetics induce autophagy by competitively disrupting the interaction between Beclin-1 and Bcl-2 or Bcl-X(L).     

Connection between induction of DNA lesions and DNA recombination/repair during Ig class switch recombination

Comment on: Kracker S, et al. Proc Natl Acad Sci USA 2010; 107:22225-30.     

Functional interplay between BRCA2/FancD1 and FancC in DNA repair

A rare hereditary disorder, Fanconi anemia (FA), is caused by mutations in an array of genes, which interact in a common FA pathway/network. These genes encode components of the FA "core" complex, a key factor FancD2, the familial breast cancer suppressor BRCA2/FancD1, and Brip1/FancJ helicase. Although BRCA2 is known to play a pivotal role in homologous recombination repair by regulating Rad51 recombinase, the precise functional relationship between BRCA2 and the other FA genes is unclear. Here we show that BRCA2-dependent chromatin loading of Rad51 after mitomycin C treatment was not compromised by disruption of FANCC or FANCD2. Rad51 and FancD2 form colocalizing subnuclear foci independently of each other. Furthermore, we created a conditional BRCA2 truncating mutation lacking the C-terminal conserved domain (CTD) (brca2DeltaCTD), and disrupted the FANCC gene in this background. The fancc/brca2DeltaCTD double mutant revealed an epistatic relationship between FANCC and BRCA2 CTD in terms of x-ray sensitivity. In contrast, levels of cisplatin sensitivity and mitomycin C-induced chromosomal aberrations were increased in fancc/brca2DeltaCTD cells relative to either single mutant. Taken together, these results indicate that FA proteins work together with BRCA2/Rad51-mediated homologous recombination in double strand break repair, whereas the FA pathway plays a role that is independent of the CTD of BRCA2 in interstrand cross-link repair. These results provide insights into the functional interplay between the classical FA pathway and BRCA2.     

Functional interaction between FOXO3a and ATM regulates DNA damage response

The maintenance of genomic stability in cells is relentlessly challenged by environmental stresses that induce DNA breaks, which activate the DNA-damage pathway mediated by ataxia-telangiectasia mutated (ATM) and its downstream mediators to control damage-induced cell-cycle checkpoints and DNA repair. Here, we show that FOXO3a interacts with ATM to promote phosphorylation of ATM at Ser 1981 and prompting its downstream mediators to form nuclear foci in response to DNA damage. Silencing FOXO3a in cells abrogates the formation of ATM-pS1981 and phospho-histone H2AX foci after DNA damage. Increasing FOXO3a in cells promotes ATM-regulated signalling, the intra-S-phase or G2-M cell-cycle checkpoints, and the repair of damaged DNA, whereas cells lacking FOXO3a did not trigger the DNA-repair mechanism after DNA damage. The carboxy-terminal domain of FOXO3a binds to the FAT domain of ATM, thereby contributing to the activation of ATM. These results suggest that ATM may be regulated directly by FOXO3a in the DNA-damage response.     

Srs2 and Sgs1-Top3 suppress crossovers during double-strand break repair in yeast

Very few gene conversions in mitotic cells are associated with crossovers, suggesting that these events are regulated. This may be important for the maintenance of genetic stability. We have analyzed the relationship between homologous recombination and crossing-over in haploid budding yeast and identified factors involved in the regulation of crossover outcomes. Gene conversions unaccompanied by a crossover appear 30 min before conversions accompanied by exchange, indicating that there are two different repair mechanisms in mitotic cells. Crossovers are rare (5%), but deleting the BLM/WRN homolog, SGS1, or the SRS2 helicase increases crossovers 2- to 3-fold. Overexpressing SRS2 nearly eliminates crossovers, whereas overexpression of RAD51 in srs2Delta cells almost completely eliminates the noncrossover recombination pathway. We suggest Sgs1 and its associated topoisomerase Top3 remove double Holliday junction intermediates from a crossover-producing repair pathway, thereby reducing crossovers. Srs2 promotes the noncrossover synthesis-dependent strand-annealing (SDSA) pathway, apparently by regulating Rad51 binding during strand exchange.     

Top3 processes recombination intermediates and modulates checkpoint activity after DNA damage

Mutation of TOP3 in Saccharomyces cerevisiae causes poor growth, hyperrecombination, and a failure to fully activate DNA damage checkpoints in S phase. Here, we report that overexpression of a dominant-negative allele of TOP3, TOP3(Y356F), which lacks the catalytic (decatenation) activity of Top3, causes impaired S-phase progression and the persistence of abnormal DNA structures (X-shaped DNA molecules) after exposure to methylmethanesulfonate. The impaired S-phase progression is due to a persistent checkpoint-mediated cell cycle delay and can be overridden by addition of caffeine. Hence, the catalytic activity of Top3 is not required for DNA damage checkpoint activation, but it is required for normal S-phase progression after DNA damage. We also present evidence that the checkpoint-mediated cell cycle delay and persistence of X-shaped DNA molecules resulting from overexpression of TOP3(Y356F) are downstream of Rad51 function. We propose that Top3 functions in S phase to both process homologous recombination intermediates and modulate checkpoint activity.     

An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase III.

XRCC1, the human gene that fully corrects the Chinese hamster ovary DNA repair mutant EM9, encodes a protein involved in the rejoining of DNA single-strand breaks that arise following treatment with alkylating agents or ionizing radiation. In this study, a cDNA minigene encoding oligohistidine-tagged XRCC1 was constructed to facilitate affinity purification of the recombinant protein. This construct, designated pcD2EHX, fully corrected the EM9 phenotype of high sister chromatid exchange, indicating that the histidine tag was not detrimental to XRCC1 activity. Affinity chromatography of extract from EM9 cells transfected with pcD2EHX resulted in the copurification of histidine-tagged XRCC1 and DNA ligase III activity. Neither XRCC1 or DNA ligase III activity was purified during affinity chromatography of extract from EM9 cells transfected with pcD2EX, a cDNA minigene that encodes untagged XRCC1, or extract from wild-type AA8 or untransfected EM9 cells. The copurification of DNA ligase III activity with histidine-tagged XRCC1 suggests that the two proteins are present in the cell as a complex. Furthermore, DNA ligase III activity was present at lower levels in EM9 cells than in AA8 cells and was returned to normal levels in EM9 cells transfected with pcD2EHX or pcD2EX. These findings indicate that XRCC1 is required for normal levels of DNA ligase III activity, and they implicate a major role for this DNA ligase in DNA base excision repair in mammalian cells.     

Requirement of Rrm3 helicase for repair of spontaneous DNA lesions in cells lacking Srs2 or Sgs1 helicase

The Rrm3 DNA helicase of Saccharomyces cerevisiae interacts with proliferating cell nuclear antigen and is required for replication fork progression through ribosomal DNA repeats and subtelomeric and telomeric DNA. Here, we show that rrm3 srs2 and rrm3 sgs1 mutants, in which two different DNA helicases have been inactivated, exhibit a severe growth defect and undergo frequent cell death. Cells lacking Rrm3 and Srs2 arrest in the G(2)/M phase of the cell cycle with 2N DNA content and frequently contain only a single nucleus. The phenotypes of rrm3 srs2 and rrm3 sgs1 mutants were suppressed by disrupting early steps of homologous recombination. These observations identify Rrm3 as a new member of a network of pathways, involving Sgs1 and Srs2 helicases and Mus81 endonuclease, suggested to act during repair of stalled replication forks.     

Homologous recombination in DNA repair and DNA damage tolerance

Homologous recombination （HR） comprises a series of interrelated pathways that function in the repair of DNA double-stranded breaks （DSBs） and interstrand crosslinks （ICLs）. In addition, recombination provides critical support for DNA replication in the recovery of stalled or broken replication forks, contributing to tolerance of DNA damage. A central core of proteins, most critically the RecA homolog Rad51, catalyzes the key reactions that typify HR： homology search and DNA strand invasion. The diverse functions of recombination are reflected in the need for context-specific factors that perform supplemental functions in conjunction with the core proteins. The inability to properly repair complex DNA damage and resolve DNA replication stress leads to genomic instability and contributes to cancer etiology. Mutations in the BRCA2 recombination gene cause predisposition to breast and ovarian cancer as well as Fanconi anemia, a cancer predisposition syndrome characterized by a defect in the repair of DNA interstrand crosslinks. The cellular functions of recombination are also germane to DNA-based treatment modalities of cancer, which target replicating cells by the direct or indirect induction of DNA lesions that are substrates for recombination pathways. This review focuses on mechanistic aspects of HR relating to DSB and ICL repair as well as replication fork support.     

Supercomplex formation between Mlh1-Mlh3 and Sgs1-Top3 heterocomplexes in meiotic yeast cells

The genome of Saccharomyces cerevisia encodes four mismatch repair MutL proteins and these proteins form three heterocomplexes: Mlh1-Mlh2, Mlh1-Mlh3, and Mlh1-Pms1. Only, the Mlh1-Mlh3 heterocomplex has been implicated specifically in promotion of meiotic crossing-over. In this report, we show that yeast Mlh3 co-immunoprecipitates with Sgs1 helicase in sporulating cells at late stage of meiotic prophase I. Sgs1, a member of the RecQ DNA helicase family, appears to form a stable complex with topoisomerase III (Top3) during meiosis. We suggest that Mlh1-Mlh3 heterocomplex may act as a molecular matchmaker to coordinate Sgs1-Top3 complex in the resolution of meiotic recombination intermediates.     

Functional interaction between the herpes simplex-1 DNA polymerase and UL42 protein

The herpes simplex virus 1 (HSV-1) UL42 protein, one of seven herpes-encoded polypeptides that are required for the replication of the HSV-1 genome, is found in a 1:1 complex with the HSV-1 DNA polymerase (Crute, J. J., and Lehman, I. R. (1989) J. Biol. Chem. 264, 19266-19270). To obtain herpes DNA polymerase free of UL42 protein, we have cloned and overexpressed the Pol gene in a recombinant baculovirus vector and purified the recombinant DNA polymerase to near homogeneity. Replication of singly primed M13mp18 single-stranded DNA by the recombinant enzyme in the presence of the herpes encoded single-stranded DNA-binding protein ICP8 yields in addition to some full-length product a distribution of intermediate length products by a quasi-processive mode of deoxynucleotide polymerization. Addition of the purified UL42 protein results in completely processive polymerization and the generation of full-length products. Similar processivity is observed with the HSV-1 DNA polymerase purified from herpes-infected Vero cells. Processive DNA replication by the DNA polymerase isolated from HSV-1-infected Vero cells or the recombinant DNA polymerase-UL42 protein complex requires that the single-stranded DNA be coated with saturating levels of ICP8. ICP8 which binds single-stranded DNA in a highly cooperative manner is presumably required to melt out regions of secondary structure in the single-stranded DNA template, thereby potentiating the processivity enhancing action of the UL42 protein.     

Rad9 plays an important role in DNA mismatch repair through physical interaction with MLH1

Rad9 is conserved from yeast to humans and plays roles in DNA repair (homologous recombination repair, and base-pair excision repair) and cell cycle checkpoint controls. It has not previously been reported whether Rad9 is involved in DNA mismatch repair (MMR). In this study, we have demonstrated that both human and mouse Rad9 interacts physically with the MMR protein MLH1. Disruption of the interaction by a single-point mutation in Rad9 leads to significantly reduced MMR activity. This disruption does not affect S/M checkpoint control and the first round of G₂/M checkpoint control, nor does it alter cell sensitivity to UV light, gamma rays or hydroxyurea. Our data indicate that Rad9 is an important factor in MMR and carries out its MMR function specifically through interaction with MLH1.     

Epistasis analysis between homologous recombination genes in Saccharomyces cerevisiae identifies multiple repair pathways for Sgs1, Mus81-Mms4 and RNase H2

The DNA repair genes SGS1 and MUS81 of Saccharomyces cerevisiae are thought to control alternative pathways for the repair of toxic recombination intermediates based on the fact that sgs1Δ mus81Δ synthetic lethality is suppressed in the absence of homologous recombination (HR). Although these genes appear to functionally overlap in yeast and other model systems, the specific pathways controlled by SGS1 and MUS81 are poorly defined. Epistasis analyses based on DNA damage sensitivity previously indicated that SGS1 functioned primarily downstream of RAD51, and that MUS81 was independent of RAD51. To further define these genetic pathways, we carried out a systematic epistasis analysis between the RAD52-epistasis group genes and SGS1, MUS81, and RNH202, which encodes a subunit of RNase H2. Based on synthetic-fitness interactions and DNA damage sensitivities, we find that RAD52 is epistatic to MUS81 but not SGS1. In contrast, RAD54, RAD55 and RAD57 are epistatic to SGS1, MUS81 and RNH202. As expected, SHU2 is epistatic to SGS1, while both SHU1 and SHU2 are epistatic to MUS81. Importantly, loss of any RNase H2 subunit on its own resulted in increased recombination using a simple marker-excision assay. RNase H2 is thus needed to maintain genome stability consistent with the sgs1Δ rnh202Δ synthetic fitness defect. We conclude that SGS1 and MUS81 act in parallel pathways downstream of RAD51 and RAD52, respectively. The data further indicate these pathways share common components and display complex interactions.