RESUMO
Bidirectionally moving DNA replication forks merge at termination sites composed of accidental or programmed DNA-protein barriers. If merging fails, then regions of unreplicated DNA can result in the breakage of DNA during mitosis, which in turn can give rise to genome instability. Despite its importance, little is known about the mechanisms that promote the final stages of fork merging in eukaryotes. Here we show that the Pif1 family DNA helicase Pfh1 plays a dual role in promoting replication fork termination. First, it facilitates replication past DNA-protein barriers, and second, it promotes the merging of replication forks. A failure of these processes in Pfh1-deficient cells results in aberrant chromosome segregation and heightened genome instability.
Assuntos
DNA Helicases/metabolismo , Replicação do DNA , Instabilidade Genômica , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Segregação de Cromossomos , DNA Helicases/genética , DNA Fúngico/genética , DNA Ribossômico/genética , Schizosaccharomyces/enzimologia , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
FANCM remodels branched DNA structures and plays essential roles in the cellular response to DNA replication stress. Here, we show that FANCM forms a conserved DNA-remodeling complex with a histone-fold heterodimer, MHF. We find that MHF stimulates DNA binding and replication fork remodeling by FANCM. In the cell, FANCM and MHF are rapidly recruited to forks stalled by DNA interstrand crosslinks, and both are required for cellular resistance to such lesions. In vertebrates, FANCM-MHF associates with the Fanconi anemia (FA) core complex, promotes FANCD2 monoubiquitination in response to DNA damage, and suppresses sister-chromatid exchanges. Yeast orthologs of these proteins function together to resist MMS-induced DNA damage and promote gene conversion at blocked replication forks. Thus, FANCM-MHF is an essential DNA-remodeling complex that protects replication forks from yeast to human.
Assuntos
DNA Helicases/metabolismo , DNA/metabolismo , Instabilidade Genômica , Histonas/metabolismo , Dobramento de Proteína , Multimerização Proteica , Sequência de Aminoácidos , Animais , Linhagem Celular , Galinhas , DNA/genética , Dano ao DNA , DNA Helicases/química , DNA Helicases/genética , Replicação do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Evolução Molecular , Proteínas de Grupos de Complementação da Anemia de Fanconi , Humanos , Dados de Sequência Molecular , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Alinhamento de Sequência , Troca de Cromátide IrmãRESUMO
The Fanconi anemia (FA) core complex promotes the tolerance/repair of DNA damage at stalled replication forks by catalyzing the monoubiquitination of FANCD2 and FANCI. Intriguingly, the core complex component FANCM also catalyzes branch migration of model Holliday junctions and replication forks in vitro. Here we have characterized the ortholog of FANCM in fission yeast Fml1 in order to understand the physiological significance of this activity. We show that Fml1 has at least two roles in homologous recombination-it promotes Rad51-dependent gene conversion at stalled/blocked replication forks and limits crossing over during mitotic double-strand break repair. In vitro Fml1 catalyzes both replication fork reversal and D loop disruption, indicating possible mechanisms by which it can fulfill its pro- and antirecombinogenic roles.
Assuntos
Quebras de DNA de Cadeia Dupla , DNA Helicases/metabolismo , Reparo do DNA , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Troca Genética , DNA Helicases/genética , Replicação do DNA , DNA Cruciforme , DNA Fúngico/química , DNA Fúngico/genética , DNA Fúngico/metabolismo , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Conversão Gênica , Genes Fúngicos , Humanos , Mutação , Recombinação Genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
During meiosis programmed DNA double-strand breaks (DSBs) are repaired by homologous recombination using the sister chromatid or the homologous chromosome (homolog) as a template. This repair results in crossover (CO) and non-crossover (NCO) recombinants. Only CO formation between homologs provides the physical linkages guiding correct chromosome segregation, which are essential to produce healthy gametes. The factors that determine the CO/NCO decision are still poorly understood. Using Schizosaccharomyces pombe as a model we show that the Rad51/Dmc1-paralog complexes Rad55-Rad57 and Rdl1-Rlp1-Sws1 together with Swi5-Sfr1 play a major role in antagonizing both the FANCM-family DNA helicase/translocase Fml1 and the RecQ-type DNA helicase Rqh1 to limit hybrid DNA formation and promote Mus81-Eme1-dependent COs. A common attribute of these protein complexes is an ability to stabilize the Rad51/Dmc1 nucleoprotein filament, and we propose that it is this property that imposes constraints on which enzymes gain access to the recombination intermediate, thereby controlling the manner in which it is processed and resolved.
Assuntos
DNA Helicases/fisiologia , Proteínas de Ligação a DNA/fisiologia , Meiose/genética , Recombinação Genética , Proteínas de Schizosaccharomyces pombe/fisiologia , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/fisiologia , Quebras de DNA de Cadeia Dupla , DNA Helicases/antagonistas & inibidores , DNA Helicases/genética , Reparo do DNA , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Endonucleases/fisiologia , Deleção de Genes , Recombinases Rec A/genética , Recombinases Rec A/fisiologia , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
Centromere proteins CENP-S and CENP-X are members of the constitutive centromere-associated network, which is a conserved group of proteins that are needed for the assembly and function of kinetochores at centromeres. Intriguingly CENP-S and CENP-X have alter egos going by the names of MHF1 (FANCM-associated histone-fold protein 1) and MHF2 respectively. In this guise they function with a DNA translocase called FANCM (Fanconi's anemia complementation group M) to promote DNA repair and homologous recombination. In the present review we discuss current knowledge of the biological roles of CENP-S and CENP-X and how their dual existence may be a common feature of CCAN (constitutive centromere-associated network) proteins.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Recombinação Genética , Proteínas Supressoras de Tumor/metabolismo , DNA/genética , DNA/metabolismo , HumanosRESUMO
The F-box DNA helicase Fbh1 constrains homologous recombination in vegetative cells, most likely through an ability to displace the Rad51 recombinase from DNA. Here, we provide the first evidence that Fbh1 also serves a vital meiotic role in fission yeast to promote normal chromosome segregation. In the absence of Fbh1, chromosomes remain entangled or segregate unevenly during meiosis, and genetic and cytological data suggest that this results in part from a failure to efficiently dismantle Rad51 nucleofilaments that form during meiotic double-strand break repair.
Assuntos
Segregação de Cromossomos , DNA Helicases/fisiologia , Proteínas F-Box/fisiologia , Meiose/genética , Rad51 Recombinase/análise , Proteínas de Schizosaccharomyces pombe/análise , Proteínas de Schizosaccharomyces pombe/fisiologia , Schizosaccharomyces/genética , Quebras de DNA de Cadeia Dupla , DNA Helicases/análise , DNA Helicases/genética , Reparo do DNA , DNA Fúngico/análise , Proteínas de Ligação a DNA/genética , Proteínas F-Box/análise , Proteínas F-Box/genética , Conversão Gênica , Deleção de Genes , Proteínas Nucleares/análise , Recombinação Genética , Schizosaccharomyces/fisiologia , Proteínas de Schizosaccharomyces pombe/genética , Esporos Fúngicos/crescimento & desenvolvimentoRESUMO
Most DNA double-strand breaks (DSBs) in S- and G2-phase cells are repaired accurately by Rad51-dependent sister chromatid recombination. However, a minority give rise to gross chromosome rearrangements (GCRs), which can result in disease/death. What determines whether a DSB is repaired accurately or inaccurately is currently unclear. We provide evidence that suggests that perturbing replication by a non-programmed protein-DNA replication fork barrier results in the persistence of replication intermediates (most likely regions of unreplicated DNA) into mitosis, which results in anaphase bridge formation and ultimately to DNA breakage. However, unlike previously characterised replication-associated DSBs, these breaks are repaired mainly by Rad51-independent processes such as single-strand annealing, and are therefore prone to generate GCRs. These data highlight how a replication-associated DSB can be predisposed to give rise to genome rearrangements in eukaryotes.
Assuntos
Quebras de DNA de Cadeia Dupla , Replicação do DNA , Recombinação Genética , Anáfase/genética , Deleção Cromossômica , DNA/ultraestrutura , DNA Helicases/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Repressores Lac/metabolismo , Mitose , Mutação , Regiões Operadoras Genéticas , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
It is thought that many of the simple and complex genomic rearrangements associated with congenital diseases and cancers stem from mistakes made during the restart of collapsed replication forks by recombination enzymes. It is hypothesised that this recombination-mediated restart process transitions from a relatively accurate initiation phase to a less accurate elongation phase characterised by extensive template switching between homologous, homeologous and microhomologous DNA sequences. Using an experimental system in fission yeast, where fork collapse is triggered by a site-specific replication barrier, we show that ectopic recombination, associated with the initiation of recombination-dependent replication (RDR), is driven mainly by the Rad51 recombinase, whereas template switching, during the elongation phase of RDR, relies more on DNA annealing by Rad52. This finding provides both evidence and a mechanistic basis for the transition hypothesis.
Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Replicação do DNA , DNA , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Ligação a DNA/metabolismoRESUMO
Meiotic recombination enhances genetic diversity as well as ensures proper segregation of homologous chromosomes, requiring Spo11-initiated double-strand breaks (DSBs). DNA deaminases act on regions of single-stranded DNA and deaminate cytosine to uracil (dU). In the immunoglobulin locus, this lesion will initiate point mutations, gene conversion, and DNA recombination. To begin to delineate the effect of induced base lesions on meiosis, we analyzed the effect of expressing DNA deaminases (activation-induced deaminase, AID, and APOBEC3C) in germ cells. We show that meiotic dU:dG lesions can partially rescue a spo11Delta phenotype in yeast and worm. In rec12 Schizosaccharomyces pombe, AID expression increased proper chromosome segregation, thereby enhancing spore viability, and induced low-frequency meiotic crossovers. Expression of AID in the germ cells of Caenorhabditis elegans spo-11 induced meiotic RAD-51 foci formation and chromosomal bivalency and segregation, as well as an increase in viability. RNAi experiments showed that this rescue was dependent on uracil DNA-glycosylase (Ung). Furthermore, unlike ionizing radiation-induced spo-11 rescue, AID expression did not induce large numbers of DSBs during the rescue. This suggests that the products of DNA deamination and base excision repair, such as uracil, an abasic site, or a single-stranded nick, are sufficient to initiate and alter meiotic recombination in uni- and multicellular organisms.
Assuntos
Segregação de Cromossomos , Citidina Desaminase/genética , Meiose/fisiologia , Recombinação Genética , Animais , Animais Geneticamente Modificados , Apoptose , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Endodesoxirribonucleases , Esterases/genética , Esterases/metabolismo , Humanos , Marcação In Situ das Extremidades Cortadas , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
Replication forks (RFs) frequently encounter barriers or lesions in template DNA that can cause them to stall and/or break. Efficient genome duplication therefore depends on multiple mechanisms that variously act to stabilize, repair, and restart perturbed RFs. Integral to at least some of these mechanisms are homologous recombination (HR) proteins, but our knowledge of how they act to ensure high-fidelity genome replication remains incomplete. To help better understand the relationship between DNA replication and HR, fission yeast strains have been engineered to contain intrachromosmal recombination substrates consisting of non-tandem direct repeats of ade6 heteroalleles. The substrates have been modified to include site-specific RF barriers within the duplication. Importantly, direct repeat recombinants appear to arise predominantly during DNA replication via sister chromatid interactions and are induced by factors that perturb RFs. Using simple plating experiments to assay recombinant formation, these strains have proved to be useful tools in monitoring the effects of impeding RFs on HR and its genetic control. The strains are available on request, and here we describe in detail how some of them can be used to determine the effect of your mutation of choice on spontaneous, DNA damage-induced, and replication block-induced recombinant formation.
Assuntos
Replicação do DNA , DNA Fúngico/biossíntese , DNA Fúngico/genética , Recombinação Genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Dano ao DNA , Genes Fúngicos , Mutagênicos/toxicidade , Mutação , Recombinação Genética/efeitos dos fármacos , Recombinação Genética/efeitos da radiação , Sequências Repetitivas de Ácido Nucleico , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/efeitos da radiação , Raios UltravioletaRESUMO
Homologous recombination helps ensure the timely completion of genome duplication by restarting collapsed replication forks. However, this beneficial function is not without risk as replication restarted by homologous recombination is prone to template switching (TS) that can generate deleterious genome rearrangements associated with diseases such as cancer. Previously we established an assay for studying TS in Schizosaccharomyces pombe (Nguyen et al., 2015). Here, we show that TS is detected up to 75 kb downstream of a collapsed replication fork and can be triggered by head-on collision between the restarted fork and RNA Polymerase III transcription. The Pif1 DNA helicase, Pfh1, promotes efficient restart and also suppresses TS. A further three conserved helicases (Fbh1, Rqh1 and Srs2) strongly suppress TS, but there is no change in TS frequency in cells lacking Fml1 or Mus81. We discuss how these factors likely influence TS.
Assuntos
Replicação do DNA/genética , Recombinação Homóloga/genética , Schizosaccharomyces/genética , Moldes Genéticos , Pareamento de Bases/genética , Mutação/genética , RNA de Transferência/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
Protein-DNA complexes can impede DNA replication and cause replication fork collapse. Whilst it is known that homologous recombination is deployed in such instances to restart replication, it is unclear how a stalled fork transitions into a collapsed fork at which recombination proteins can load. Previously we established assays in Schizosaccharomyces pombe for studying recombination induced by replication fork collapse at the site-specific protein-DNA barrier RTS1 (Nguyen et al., 2015). Here, we provide evidence that efficient recruitment/retention of two key recombination proteins (Rad51 and Rad52) to RTS1 depends on unloading of the polymerase sliding clamp PCNA from DNA by Elg1. We also show that, in the absence of Elg1, reduced recombination is partially suppressed by deleting fbh1 or, to a lesser extent, srs2, which encode known anti-recombinogenic DNA helicases. These findings suggest that PCNA unloading by Elg1 is necessary to limit Fbh1 and Srs2 activity, and thereby enable recombination to proceed.
Assuntos
Proteínas de Transporte/metabolismo , Replicação do DNA , Antígeno Nuclear de Célula em Proliferação/metabolismo , Recombinação Genética/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , DNA Fúngico/metabolismo , Fluorescência , Modelos Biológicos , Mutação/genética , Fase SRESUMO
Previously we reported that a process called inter-fork strand annealing (IFSA) causes genomic deletions during the termination of DNA replication when an active replication fork converges on a collapsed fork (Morrow et al., 2017). We also identified the FANCM-related DNA helicase Fml1 as a potential suppressor of IFSA. Here, we confirm that Fml1 does indeed suppress IFSA, and show that this function depends on its catalytic activity and ability to interact with Mhf1-Mhf2 via its C-terminal domain. Finally, a plausible mechanism of IFSA suppression is demonstrated by the finding that Fml1 can catalyse regressed fork restoration in vitro.
Assuntos
Proteínas Cromossômicas não Histona/genética , DNA Helicases/genética , Recombinação Genética , Proteínas de Schizosaccharomyces pombe/genética , Replicação do DNA/genética , Genoma Fúngico/genética , Mitose/genética , Schizosaccharomyces/genéticaRESUMO
Cells of all living organisms have evolved complex mechanisms that serve to stabilise, repair and restart stalled, blocked and broken replication forks. The heterodimeric Mus81-Eme1/Mms4 structure-specific endonuclease appears to play an important role(s) in homologous recombination-mediated processing of such perturbed forks. This enzyme has been implicated in the cleavage of stalled and blocked replication forks to initiate recombination, as well as in the processing of recombination intermediates that result from repairing damaged forks. In this review we assess the biochemical and genetic evidence for the mitotic role of Mus81-Eme1/Mms4 at replication forks and in repairing post-replication DNA damage. Mus81 appears to act when replication is impeded by genotoxins or by impairment of the replication machinery, or when arrested replication forks are not adequately protected. We discuss how its action is regulated by the S-phase cell cycle checkpoint, depending on the nature of the stalled or damaged fork. We also present a new way in which Mus81 may limit crossing over during the repair of post-replication gaps, and explore Mus81's interplay with other components of the recombination machinery, including the RecQ helicases that also play important roles in processing replication and recombination intermediates.
Assuntos
Reparo do DNA , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Animais , Quebras de DNA de Cadeia Dupla , Endodesoxirribonucleases/metabolismo , Endonucleases Flap , Humanos , Fase S , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transativadores/metabolismoRESUMO
A key step in homologous recombination is the loading of Rad51 onto single-stranded DNA to form a nucleoprotein filament that promotes homologous DNA pairing and strand exchange. Mediator proteins, such as Rad52 and Rad55-Rad57, are thought to aid filament assembly by overcoming an inhibitory effect of the single-stranded-DNA-binding protein replication protein A. Here we show that mediator proteins are also required to enable fission yeast Rad51 (called Rhp51) to function in the presence of the F-box DNA helicase Fbh1. In particular, we show that the critical function of Rad22 (an orthologue of Rad52) in promoting Rhp51-dependent recombination and DNA repair can be mostly circumvented by deleting fbh1. Similarly, the reduced growth/viability and DNA damage sensitivity of an fbh1(-) mutant are variously suppressed by deletion of any one of the mediators Rad22, Rhp55, and Swi5. From these data we propose that Rhp51 action is controlled through an interplay between Fbh1 and the mediator proteins. Colocalization of Fbh1 with Rhp51 damage-induced foci suggests that this interplay occurs at the sites of nucleoprotein filament assembly. Furthermore, analysis of different fbh1 mutant alleles suggests that both the F-box and helicase activities of Fbh1 contribute to controlling Rhp51.
Assuntos
DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas F-Box/metabolismo , Recombinação Genética/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Núcleo Celular/química , Núcleo Celular/metabolismo , Segregação de Cromossomos/genética , Segregação de Cromossomos/fisiologia , Dano ao DNA/genética , DNA Helicases/análise , DNA Helicases/genética , Reparo do DNA/genética , Reparo do DNA/fisiologia , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/genética , Proteínas F-Box/genética , Deleção de Genes , Mutação , Rad51 Recombinase , Recombinação Genética/fisiologia , Schizosaccharomyces/enzimologia , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/análise , Proteínas de Schizosaccharomyces pombe/genética , Supressão GenéticaRESUMO
Problems that arise during DNA replication can drive genomic alterations that are instrumental in the development of cancers and many human genetic disorders. Replication fork barriers are a commonly encountered problem, which can cause fork collapse and act as hotspots for replication termination. Collapsed forks can be rescued by homologous recombination, which restarts replication. However, replication restart is relatively slow and, therefore, replication termination may frequently occur by an active fork converging on a collapsed fork. We find that this type of non-canonical fork convergence in fission yeast is prone to trigger deletions between repetitive DNA sequences via a mechanism we call Inter-Fork Strand Annealing (IFSA) that depends on the recombination proteins Rad52, Exo1 and Mus81, and is countered by the FANCM-related DNA helicase Fml1. Based on our findings, we propose that IFSA is a potential threat to genomic stability in eukaryotes.
Assuntos
Pareamento de Bases , Replicação do DNA , Recombinação Homóloga , Schizosaccharomyces/genética , Deleção de Sequência , DNA Helicases/metabolismo , Instabilidade Genômica , Recombinases/metabolismo , Schizosaccharomyces/enzimologiaRESUMO
In budding yeast most Rad51-dependent and -independent recombination depends on Rad52. In contrast, its homologue in fission yeast, Rad22, was assumed to play a less critical role possibly due to functional redundancy with another Rad52-like protein Rti1. We show here that this is not the case. Rad22 like Rad52 plays a central role in recombination being required for both Rhp51-dependent and -independent events. Having established this we proceed to investigate the involvement of the Mus81-Eme1 endonuclease in these pathways. Mus81 plays a relatively minor role in the Rhp51-dependent repair of DNA damage induced by ultraviolet light. In contrast Mus81 has a key role in the Rad22-dependent (Rhp51-independent) repair of damage induced by camptothecin, hydroxyurea and methyl-methanesulfonate. Furthermore, spontaneous intrachromosomal recombination that gives rise to deletion recombinants is impaired in a mus81 mutant. From these data we propose that a Rad22-Mus81-dependent (Rhp51-independent) pathway is an important mechanism for the repair of DNA damage in fission yeast. Consistent with this we show that in vitro Rad22 can promote strand invasion to form a D-loop that can be cleaved by Mus81.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , DNA Fúngico/química , DNA Fúngico/genética , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Endonucleases/deficiência , Endonucleases/genética , Modelos Genéticos , Mutação/genética , Conformação de Ácido Nucleico , Rad51 Recombinase , Recombinação Genética/genética , Sequências Repetitivas de Ácido Nucleico/genética , Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
One of the most frequent lesions formed in cellular DNA are abasic (apurinic/apyrimidinic, AP) sites that are both cytotoxic and mutagenic, and must be removed efficiently to maintain genetic stability. It is generally believed that the repair of AP sites is initiated by the AP endonucleases; however, an alternative pathway seems to prevail in Schizosaccharomyces pombe. A mutant lacking the DNA glycosylase/AP lyase Nth1 is very sensitive to the alkylating agent methyl methanesulfonate (MMS), suggesting a role for Nth1 in base excision repair (BER) of alkylation damage. Here, we have further evaluated the role of Nth1 and the second putative S.pombe AP endonuclease Apn2, in abasic site repair. The deletion of the apn2 open reading frame dramatically increased the sensitivity of the yeast cells to MMS, also demonstrating that the Apn2 has an important function in the BER pathway. The deletion of nth1 in the apn2 mutant strain partially relieves the MMS sensitivity of the apn2 single mutant, indicating that the Apn2 and Nth1 act in the same pathway for the repair of abasic sites. Analysis of the AP site cleavage in whole cell extracts of wild-type and mutant strains showed that the AP lyase activity of Nth1 represents the major AP site incision activity in vitro. Assays with DNA substrates containing base lesions removed by monofunctional DNA glycosylases Udg and MutY showed that Nth1 will also cleave the abasic sites formed by these enzymes and thus act downstream of these enzymes in the BER pathway. We suggest that the main function of Apn2 in BER is to remove the resulting 3'-blocking termini following AP lyase cleavage by Nth1.
Assuntos
DNA Glicosilases/fisiologia , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/fisiologia , Complexos Multienzimáticos/fisiologia , Proteínas de Schizosaccharomyces pombe/fisiologia , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Análise Mutacional de DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Complexos Multienzimáticos/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
DNA double-strand break (DSB) repair by homologous recombination (HR) involves resection of the break to expose a 3' single-stranded DNA tail. In budding yeast, resection occurs in two steps: initial short-range resection, performed by Mre11-Rad50-Xrs2 and Sae2; and long-range resection catalysed by either Exo1 or Sgs1-Dna2. Here we use genetic assays to investigate the importance of Exo1 and the Sgs1 homologue Rqh1 for DNA repair and promotion of direct repeat recombination in the fission yeast Schizosaccharomyces pombe. We find that Exo1 and Rqh1 function in alternative redundant pathways for promoting survival following replication fork breakage. Exo1 promotes replication fork barrier-induced direct repeat recombination but intriguingly limits recombination induced by fork breakage. Direct repeat recombination induced by ultraviolet light depends on either Exo1 or Rqh1. Finally, we show that Rqh1 plays a major role in limiting Exo1-dependent direct repeat recombination induced by replication fork stalling but only a minor role in constraining recombination induced by fork breakage. The implications of our findings are discussed in the context of the benefits that long-range resection may bring to processing perturbed replication forks.