ABSTRACT
When replication forks stall at damaged bases or upon nucleotide depletion, the intra-S phase checkpoint ensures they are stabilized and can restart. In intra-S checkpoint-deficient budding yeast, stalling forks collapse, and â¼10% form pathogenic chicken foot structures, contributing to incomplete replication and cell death (Lopes et al., 2001; Sogo et al., 2002; Tercero and Diffley, 2001). Using fission yeast, we report that the Cds1(Chk2) effector kinase targets Dna2 on S220 to regulate, both in vivo and in vitro, Dna2 association with stalled replication forks in chromatin. We demonstrate that Dna2-S220 phosphorylation and the nuclease activity of Dna2 are required to prevent fork reversal. Consistent with this, Dna2 can efficiently cleave obligate precursors of fork regression-regressed leading or lagging strands-on model replication forks. We propose that Dna2 cleavage of regressed nascent strands prevents fork reversal and thus stabilizes stalled forks to maintain genome stability during replication stress.
Subject(s)
DNA Replication , Flap Endonucleases/metabolism , S Phase Cell Cycle Checkpoints , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/cytology , Schizosaccharomyces/metabolism , Checkpoint Kinase 2 , Epistasis, Genetic , Genomic Instability , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Schizosaccharomyces/geneticsABSTRACT
Chromosomal instability (CIN) drives cell-to-cell heterogeneity, and the development of genetic diseases, including cancer. Impaired homologous recombination (HR) has been implicated as a major driver of CIN, however, the underlying mechanism remains unclear. Using a fission yeast model system, we establish a common role for HR genes in suppressing DNA double-strand break (DSB)-induced CIN. Further, we show that an unrepaired single-ended DSB arising from failed HR repair or telomere loss is a potent driver of widespread CIN. Inherited chromosomes carrying a single-ended DSB are subject to cycles of DNA replication and extensive end-processing across successive cell divisions. These cycles are enabled by Cullin 3-mediated Chk1 loss and checkpoint adaptation. Subsequent propagation of unstable chromosomes carrying a single-ended DSB continues until transgenerational end-resection leads to fold-back inversion of single-stranded centromeric repeats and to stable chromosomal rearrangements, typically isochromosomes, or to chromosomal loss. These findings reveal a mechanism by which HR genes suppress CIN and how DNA breaks that persist through mitotic divisions propagate cell-to-cell heterogeneity in the resultant progeny.
Subject(s)
Schizosaccharomyces , Humans , Chromosomal Instability , DNA Breaks, Double-Stranded , DNA Repair , Homologous Recombination , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolismABSTRACT
The Smc5/6 complex plays an essential role in the resolution of recombination intermediates formed during mitosis or meiosis, or as a result of the cellular response to replication stress. It also functions as a restriction factor preventing viral replication. Here, we report the cryogenic EM (cryo-EM) structure of the six-subunit budding yeast Smc5/6 holo-complex, reconstituted from recombinant proteins expressed in insect cells - providing both an architectural overview of the entire complex and an understanding of how the Nse1/3/4 subcomplex binds to the hetero-dimeric SMC protein core. In addition, we demonstrate that a region within the head domain of Smc5, equivalent to the 'W-loop' of Smc4 or 'F-loop' of Smc1, mediates an important interaction with Nse1. Notably, mutations that alter the surface-charge profile of the region of Nse1 which accepts the Smc5-loop, lead to a slow-growth phenotype and a global reduction in the chromatin-associated fraction of the Smc5/6 complex, as judged by single molecule localisation microscopy experiments in live yeast. Moreover, when taken together, our data indicates functional equivalence between the structurally unrelated KITE and HAWK accessory subunits associated with SMC complexes.
Subject(s)
Cell Cycle Proteins , Saccharomyces cerevisiae Proteins , Cell Cycle Proteins/metabolism , Nuclear Proteins/metabolism , Cryoelectron Microscopy , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolismABSTRACT
DNA replication is dramatically slowed down under replication stress. The regulation of replication speed is a conserved response in eukaryotes and, in fission yeast, requires the checkpoint kinases Rad3ATR and Cds1Chk2 However, the underlying mechanism of this checkpoint regulation remains unresolved. Here, we report that the Rad3ATR-Cds1Chk2 checkpoint directly targets the Cdc45-MCM-GINS (CMG) replicative helicase under replication stress. When replication forks stall, the Cds1Chk2 kinase directly phosphorylates Cdc45 on the S275, S322, and S397 residues, which significantly reduces CMG helicase activity. Furthermore, in cds1Chk2 -mutated cells, the CMG helicase and DNA polymerases are physically separated, potentially disrupting replisomes and collapsing replication forks. This study demonstrates that the intra-S phase checkpoint directly regulates replication elongation, reduces CMG helicase processivity, prevents CMG helicase delinking from DNA polymerases, and therefore helps preserve the integrity of stalled replisomes and replication forks.
Subject(s)
DNA Replication , DNA-Directed DNA Polymerase , Multienzyme Complexes , S Phase Cell Cycle Checkpoints , Schizosaccharomyces/metabolism , Alleles , DNA Helicases/metabolism , DNA Replication/drug effects , DNA-Directed DNA Polymerase/metabolism , Hydroxyurea/pharmacology , Models, Biological , Multienzyme Complexes/metabolism , Multiprotein Complexes/metabolism , Mutation/genetics , Phosphorylation/drug effects , S Phase Cell Cycle Checkpoints/drug effects , Schizosaccharomyces/drug effects , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/metabolismABSTRACT
The multi-component Smc5/6 complex plays a critical role in the resolution of recombination intermediates formed during mitosis and meiosis, and in the cellular response to replication stress. Using recombinant proteins, we have reconstituted a series of defined Saccharomyces cerevisiae Smc5/6 complexes, visualised them by negative stain electron microscopy, and tested their ability to function as an ATPase. We find that only the six protein 'holo-complex' is capable of turning over ATP and that its activity is significantly increased by the addition of double-stranded DNA to reaction mixes. Furthermore, stimulation is wholly dependent on functional ATP-binding pockets in both Smc5 and Smc6. Importantly, we demonstrate that budding yeast Nse5/6 acts as a negative regulator of Smc5/6 ATPase activity, binding to the head-end of the complex to suppress turnover, irrespective of the DNA-bound status of the complex.
Subject(s)
Adenosine Triphosphatases/metabolism , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/genetics , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Cell Cycle Proteins/ultrastructure , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/ultrastructure , DNA/metabolism , Escherichia coli/metabolism , Microscopy, Electron, Transmission , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/ultrastructureABSTRACT
The healing of broken chromosomes by de novo telomere addition, while a normal developmental process in some organisms, has the potential to cause extensive loss of heterozygosity, genetic disease, or cell death. However, it is unclear how de novo telomere addition (dnTA) is regulated at DNA double-strand breaks (DSBs). Here, using a non-essential minichromosome in fission yeast, we identify roles for the HR factors Rqh1 helicase, in concert with Rad55, in suppressing dnTA at or near a DSB. We find the frequency of dnTA in rqh1Δ rad55Δ cells is reduced following loss of Exo1, Swi5 or Rad51. Strikingly, in the absence of the distal homologous chromosome arm dnTA is further increased, with nearly half of the breaks being healed in rqh1Δ rad55Δ or rqh1Δ exo1Δ cells. These findings provide new insights into the genetic context of highly efficient dnTA within HR intermediates, and how such events are normally suppressed to maintain genome stability.
Subject(s)
DNA Helicases/genetics , DNA-Binding Proteins/genetics , Recombinational DNA Repair/genetics , Schizosaccharomyces pombe Proteins/genetics , Telomere/genetics , Chromosomes, Fungal/genetics , DNA Breaks, Double-Stranded , Exodeoxyribonucleases/genetics , Gene Expression Regulation, Fungal/genetics , Genome, Fungal/genetics , Genomic Instability/genetics , Loss of Heterozygosity/genetics , Rad51 Recombinase/genetics , Schizosaccharomyces/geneticsABSTRACT
Of the three structural maintenance of chromosome (SMC) complexes, two directly regulate chromosome dynamics. The third, Smc5/6, functions mainly in homologous recombination and in completing DNA replication. The literature suggests that Smc5/6 coordinates DNA repair, in part through post-translational modification of uncharacterized target proteins that can dictate their subcellular localization, and that Smc5/6 also functions to establish DNA-damage-dependent cohesion. A nucleolar-specific Smc5/6 function has been proposed because Smc5/6 yeast mutants display penetrant phenotypes of ribosomal DNA (rDNA) instability. rDNA repeats are replicated unidirectionally. Here, we propose that unidirectional replication, combined with global Smc5/6 functions, can explain the apparent rDNA specificity.
Subject(s)
Cell Cycle Proteins/physiology , DNA Repair , DNA Replication , Gene Expression Regulation , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA Damage , DNA, Ribosomal/metabolism , Humans , Models, Biological , Models, Genetic , Nuclear Proteins/metabolism , Recombination, Genetic , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/physiology , CohesinsABSTRACT
Impediments to DNA replication are known to induce gross chromosomal rearrangements (GCRs) and copy-number variations (CNVs). GCRs and CNVs underlie human genomic disorders and are a feature of cancer. During cancer development, environmental factors and oncogene-driven proliferation promote replication stress. Resulting GCRs and CNVs are proposed to contribute to cancer development and therapy resistance. When stress arrests replication, the replisome remains associated with the fork DNA (stalled fork) and is protected by the inter-S-phase checkpoint. Stalled forks efficiently resume when the stress is relieved. However, if the polymerases dissociate from the fork (fork collapse) or the fork structure breaks (broken fork), replication restart can proceed either by homologous recombination or microhomology-primed re-initiation. Here we ascertain the consequences of replication with a fork restarted by homologous recombination in fission yeast. We identify a new mechanism of chromosomal rearrangement through the observation that recombination-restarted forks have a considerably high propensity to execute a U-turn at small inverted repeats (up to 1 in 40 replication events). We propose that the error-prone nature of restarted forks contributes to the generation of GCRs and gene amplification in cancer, and to non-recurrent CNVs in genomic disorders.
Subject(s)
Chromosome Inversion/genetics , DNA Replication/genetics , Inverted Repeat Sequences/genetics , Models, Genetic , Recombination, Genetic/genetics , Schizosaccharomyces/genetics , DNA Copy Number Variations/genetics , DNA, Fungal/genetics , DNA, Ribosomal/genetics , Genes, Fungal/genetics , Neoplasms/genetics , Saccharomyces cerevisiae/geneticsABSTRACT
The cellular response to DNA damage during S-phase regulates a complicated network of processes, including cell-cycle progression, gene expression, DNA replication kinetics, and DNA repair. In fission yeast, this S-phase DNA damage response (DDR) is coordinated by two protein kinases: Rad3, the ortholog of mammalian ATR, and Cds1, the ortholog of mammalian Chk2. Although several critical downstream targets of Rad3 and Cds1 have been identified, most of their presumed targets are unknown, including the targets responsible for regulating replication kinetics and coordinating replication and repair. To characterize targets of the S-phase DDR, we identified proteins phosphorylated in response to methyl methanesulfonate (MMS)-induced S-phase DNA damage in wild-type, rad3∆, and cds1∆ cells by proteome-wide mass spectrometry. We found a broad range of S-phase-specific DDR targets involved in gene expression, stress response, regulation of mitosis and cytokinesis, and DNA replication and repair. These targets are highly enriched for proteins required for viability in response to MMS, indicating their biological significance. Furthermore, the regulation of these proteins is similar in fission and budding yeast, across 300 My of evolution, demonstrating a deep conservation of S-phase DDR targets and suggesting that these targets may be critical for maintaining genome stability in response to S-phase DNA damage across eukaryotes.
Subject(s)
DNA Damage , S Phase , Schizosaccharomyces/genetics , Checkpoint Kinase 2/genetics , Checkpoint Kinase 2/metabolism , DNA Damage/drug effects , Gene Expression Regulation, Fungal/drug effects , Genomic Instability/drug effects , Methyl Methanesulfonate/toxicity , S Phase/drug effects , Schizosaccharomyces/cytology , Schizosaccharomyces/drug effects , Schizosaccharomyces/enzymology , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/metabolismABSTRACT
Template switching induced by stalled replication forks has recently been proposed to underlie complex genomic rearrangements. However, the resulting models are not supported by robust physical evidence. Here, we analyzed replication and recombination intermediates in a well-defined fission yeast system that blocks replication forks. We show that, in response to fork arrest, chromosomal rearrangements result from Rad52-dependent nascent strand template exchange occurring during fork restart. This template exchange occurs by both Rad51-dependent and -independent mechanisms. We demonstrate that Rqh1, the BLM homolog, limits Rad51-dependent template exchange without affecting fork restart. In contrast, we report that the Srs2 helicase promotes both fork restart and template exchange. Our data demonstrate that template exchange occurs during recombination-dependent fork restart at the expense of genome rearrangements.
Subject(s)
DNA Replication/physiology , DNA, Fungal/biosynthesis , Genome, Fungal/physiology , Recombination, Genetic/physiology , Schizosaccharomyces/metabolism , DNA Helicases/genetics , DNA Helicases/metabolism , DNA, Fungal/genetics , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , Rad52 DNA Repair and Recombination Protein/genetics , Rad52 DNA Repair and Recombination Protein/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/metabolismABSTRACT
Gene amplification plays important roles in the progression of cancer and contributes to acquired drug resistance during treatment. Amplification can initiate via dicentric palindromic chromosome production and subsequent breakage-fusion-bridge cycles. Here we show that, in fission yeast, acentric and dicentric palindromic chromosomes form by homologous recombination protein-dependent fusion of nearby inverted repeats, and that these fusions occur frequently when replication forks arrest within the inverted repeats. Genetic and molecular analyses suggest that these acentric and dicentric palindromic chromosomes arise not by previously described mechanisms, but by a replication template exchange mechanism that does not involve a DNA double-strand break. We thus propose an alternative mechanism for the generation of palindromic chromosomes dependent on replication fork arrest at closely spaced inverted repeats.
Subject(s)
Chromosomes, Fungal/genetics , DNA Replication/genetics , DNA, Fungal/genetics , Inverted Repeat Sequences/genetics , Schizosaccharomyces/geneticsABSTRACT
BRCT-containing protein 1 (Brc1) is a multi-BRCT (BRCA1 carboxyl terminus) domain protein in Schizosaccharomyces pombe that is required for resistance to chronic replicative stress, but whether this reflects a repair or replication defect is unknown and the subject of this study. We show that brc1Δ cells are significantly delayed in recovery from replication pausing, though this does not activate a DNA damage checkpoint. DNA repair and recombination protein Rad52 is a homologous recombination protein that loads the Rad51 recombinase at resected double-stranded DNA (dsDNA) breaks and is also recruited to stalled replication forks, where it may stabilize structures through its strand annealing activity. Rad52 is required for the viability of brc1Δ cells, and brc1Δ cells accumulate Rad52 foci late in S phase that are potentiated by replication stress. However, these foci contain the single-stranded DNA (ssDNA) binding protein RPA, but not Rad51 or γH2A. Further, these foci are not associated with increased recombination between repeated sequences, or increased post-replication repair. Thus, these Rad52 foci do not represent sites of recombination. Following the initiation of DNA replication, the induction of these foci by replication stress is suppressed by defects in origin recognition complex (ORC) function, which is accompanied by loss of viability and severe mitotic defects. This suggests that cells lacking Brc1 undergo an ORC-dependent rescue of replication stress, presumably through the firing of dormant origins, and this generates RPA-coated ssDNA and recruits Rad52. However, as Rad51 is not recruited, and the checkpoint effector kinase Chk1 is not activated, these structures must not contain the unprotected primer ends found at sites of DNA damage that are required for recombination and checkpoint activation.
Subject(s)
DNA Replication , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/cytology , Schizosaccharomyces/metabolism , Stress, Physiological , DNA Replication/drug effects , DNA, Ribosomal/metabolism , Hydroxyurea/pharmacology , Mutagenesis/drug effects , Mutagenesis/genetics , Mutation/genetics , Origin Recognition Complex/metabolism , Rad52 DNA Repair and Recombination Protein/metabolism , Recombination, Genetic/genetics , Replication Protein A/metabolism , S Phase/drug effects , Schizosaccharomyces/drug effects , Signal Transduction/drug effects , Stress, Physiological/drug effectsABSTRACT
In the original publication [...].
ABSTRACT
The Smc5/6 structural maintenance of chromosomes complex is required for efficient homologous recombination (HR). Defects in Smc5/6 result in chromosome mis-segregation and fragmentation. By characterising two Schizosaccharomyces pombe smc6 mutants, we define two separate functions for Smc5/6 in HR. The first represents the previously described defect in processing recombination-dependent DNA intermediates when replication forks collapse, which leads to increased rDNA recombination. The second novel function defines Smc5/6 as a positive regulator of recombination in the rDNA and correlates mechanistically with a requirement to load RPA and Rad52 onto chromatin genome-wide when replication forks are stably stalled by nucleotide depletion. Rad52 is required for all HR repair, but Rad52 loading in response to replication fork stalling is unexpected and does not correlate with damage-induced foci. We propose that Smc5/6 is required to maintain stalled forks in a stable recombination-competent conformation primed for replication restart.
Subject(s)
Cell Cycle Proteins/physiology , Chromosomal Proteins, Non-Histone/physiology , DNA Replication/physiology , Schizosaccharomyces pombe Proteins/physiology , Schizosaccharomyces/physiology , Cell Cycle Proteins/genetics , Chromatin/genetics , Chromatin/physiology , Chromosomal Proteins, Non-Histone/genetics , DNA Damage , DNA Replication/genetics , Mutation , Nucleic Acid Conformation , Rad52 DNA Repair and Recombination Protein/genetics , Rad52 DNA Repair and Recombination Protein/metabolism , Recombination, Genetic , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/geneticsABSTRACT
DNA DSBs (double-strand breaks) represent a critical lesion for a cell, with misrepair being potentially as harmful as lack of repair. In mammalian cells, DSBs are predominantly repaired by non-homologous end-joining or homologous recombination. The kinetics of repair of DSBs can differ widely, and recent studies have shown that the higher-order chromatin structure can dramatically affect the pathway utilized, the rate of repair and the genetic factors required for repair. Studies of the repair of DSBs arising within heterochromatic DNA regions have provided insight into the constraints that higher-order chromatin structure poses on repair and the processing that is uniquely required for the repair of such DSBs. In the present paper, we provide an overview of our current understanding of the process of heterochromatic DSB repair in mammalian cells and consider the evolutionary conservation of the processes.
Subject(s)
DNA Breaks, Double-Stranded , DNA Repair , Heterochromatin/metabolism , Animals , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins/metabolism , Chromatin Assembly and Disassembly , DNA Helicases/metabolism , DNA-Binding Proteins/metabolism , Euchromatin/metabolism , Histones/metabolism , Humans , Mi-2 Nucleosome Remodeling and Deacetylase Complex , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Repressor Proteins/metabolism , Tripartite Motif-Containing Protein 28 , Tumor Suppressor Proteins/metabolismABSTRACT
Structural Maintenance of Chromosomes (SMC) complexes are important for many aspects of the chromosomal organization. Unlike cohesin and condensin, the SMC5/6 complex contains a variant RING domain carried by its Nse1 subunit. RING domains are characteristic for ubiquitin ligases, and human NSE1 has been shown to possess ubiquitin-ligase activity in vitro. However, other studies were unable to show such activity. Here, we confirm Nse1 ubiquitin-ligase activity using purified Schizosaccharomyces pombe proteins. We demonstrate that the Nse1 ligase activity is stimulated by Nse3 and Nse4. We show that Nse1 specifically utilizes Ubc13/Mms2 E2 enzyme and interacts directly with ubiquitin. We identify the Nse1 mutation (R188E) that specifically disrupts its E3 activity and demonstrate that the Nse1-dependent ubiquitination is particularly important under replication stress. Moreover, we determine Nse4 (lysine K181) as the first known SMC5/6-associated Nse1 substrate. Interestingly, abolition of Nse4 modification at K181 leads to suppression of DNA-damage sensitivity of other SMC5/6 mutants. Altogether, this study brings new evidence for Nse1 ubiquitin ligase activity, significantly advancing our understanding of this enigmatic SMC5/6 function.
Subject(s)
Carrier Proteins/metabolism , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Ligases/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Ubiquitin/metabolism , Ubiquitination/immunology , HumansABSTRACT
The essential Smc5/6 complex is required in response to replication stress and is best known for ensuring the fidelity of homologous recombination. Using single-molecule tracking in live fission yeast to investigate Smc5/6 chromatin association, we show that Smc5/6 is chromatin associated in unchallenged cells and this depends on the non-SMC protein Nse6. We define a minimum of two Nse6-dependent sub-pathways, one of which requires the BRCT-domain protein Brc1. Using defined mutants in genes encoding the core Smc5/6 complex subunits, we show that the Nse3 double-stranded DNA binding activity and the arginine fingers of the two Smc5/6 ATPase binding sites are critical for chromatin association. Interestingly, disrupting the single-stranded DNA (ssDNA) binding activity at the hinge region does not prevent chromatin association but leads to elevated levels of gross chromosomal rearrangements during replication restart. This is consistent with a downstream function for ssDNA binding in regulating homologous recombination.
Subject(s)
Cell Cycle Proteins/metabolism , Chromatin/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/metabolism , Single Molecule ImagingABSTRACT
In eukaryotes, three pairs of structural-maintenance-of-chromosome (SMC) proteins are found in conserved multisubunit protein complexes required for chromosomal organization. Cohesin, the Smc1/3 complex, mediates sister chromatid cohesion while two condensin complexes containing Smc2/4 facilitate chromosome condensation. Smc5/6 scaffolds an essential complex required for homologous recombination repair. We have examined the response of smc6 mutants to the inhibition of DNA replication. We define homologous recombination-dependent and -independent functions for Smc6 during replication inhibition and provide evidence for a Rad60-independent function within S phase, in addition to a Rad60-dependent function following S phase. Both genetic and physical data show that when forks collapse (i.e., are not stabilized by the Cds1Chk2 checkpoint), Smc6 is required for the effective repair of resulting lesions but not for the recruitment of recombination proteins. We further demonstrate that when the Rad60-dependent, post-S-phase Smc6 function is compromised, the resulting recombination-dependent DNA intermediates that accumulate following release from replication arrest are not recognized by the G2/M checkpoint.
Subject(s)
Cell Cycle Proteins/physiology , Chromosomal Proteins, Non-Histone/physiology , DNA Repair/genetics , DNA Replication/genetics , DNA, Fungal/biosynthesis , Schizosaccharomyces pombe Proteins/physiology , Schizosaccharomyces/genetics , Cell Cycle/drug effects , Cell Cycle/genetics , Cell Cycle Proteins/genetics , Chromosomal Proteins, Non-Histone/genetics , DNA Damage/genetics , Hydroxyurea/pharmacology , Mutation , Recombination, Genetic , Schizosaccharomyces/cytology , Schizosaccharomyces/growth & development , Schizosaccharomyces pombe Proteins/geneticsABSTRACT
Smc5/6 is a structural maintenance of chromosomes complex, related to the cohesin and condensin complexes. Recent studies implicate Smc5/6 as being essential for homologous recombination. Each gene is essential, but hypomorphic alleles are defective in the repair of a diverse array of lesions. A particular allele of smc6 (smc6-74) is suppressed by overexpression of Brc1, a six-BRCT domain protein that is required for DNA repair during S-phase. This suppression requires the postreplication repair (PRR) protein Rhp18 and the structure-specific endonucleases Slx1/4 and Mus81/Eme1. However, we show here that the contribution of Rhp18 is via a novel pathway that is independent of PCNA ubiquitination and PRR. Moreover, we identify Exo1 as an additional nuclease required for Brc1-mediated suppression of smc6-74, independent of mismatch repair. Further, the Apn2 endonuclease is required for the viability of smc6 mutants without extrinsic DNA damage, although this is not due to a defect in base excision repair. Several nucleotide excision repair genes are similarly shown to ensure viability of smc6 mutants. The requirement for excision factors for the viability of smc6 mutants is consistent with an inability to respond to spontaneous lesions by Smc5/6-dependent recombination.
Subject(s)
Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , Base Sequence , DNA Repair/genetics , DNA, Fungal/genetics , DNA-(Apurinic or Apyrimidinic Site) Lyase/genetics , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , Exodeoxyribonucleases/genetics , Exodeoxyribonucleases/metabolism , Genes, Fungal , Mutation , Recombination, Genetic , Schizosaccharomyces/growth & developmentABSTRACT
DNA is labile and constantly subject to damage. In addition to external mutagens, DNA is continuously damaged by the aqueous environment, cellular metabolites and is prone to strand breakage during replication. Cell duplication is orchestrated by the cell division cycle and specific DNA structures are processed differently depending on where in the cell cycle they are detected. This is often because a specific structure is physiological in one context, for example during DNA replication, while indicating a potentially pathological event in another, such as interphase or mitosis. Thus, contextualising the biochemical entity with respect to cell cycle progression provides information necessary to appropriately regulate DNA processing activities. We review the links between DNA repair and cell cycle context, drawing together recent advances.