Nse1-dependent recruitment of Smc5/6 to lesion-containing loci contributes to the repair defects of mutant complexes

Tapia-Alveal, Claudia; O’Connell, Matthew J.

doi:10.1091/mbc.e11-03-0272

Cited by 19 publications

(37 citation statements)

References 73 publications

(144 reference statements)

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“…Alternatively, the pathway can be channeled into a second arm, in which the mono-ubiquitylation of PCNA is converted to polyubiquitylation with K63 linkages, catalyzed by the Ubc13-Mms2 complex (Lee and Myung, 2008). This arm of the pathway utilizes Rad5 and the HR machinery, specifically Rad55/Rad57, to invade the other nascent strand of DNA and replicate off this template to accomplish error-free lesion bypass (Tapia-Alveal and O'Connell, 2011;Vanoli et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

“…Similar to the cohesin and condensin complexes, Smc5-Smc6 is required for accurate chromosome segregation (Hirano, 2006;Outwin et al, 2009). Although many Smc5-Smc6 mutants show defects in recombinational repair (Ampatzidou et al, 2006;Andrews et al, 2005;Morikawa et al, 2004;Pebernard et al, 2004;Verkade et al, 1999), recent evidence suggests that this defect is due to the recruitment of dysfunctional complexes to lesions, rather than an absolute requirement for Smc5-Smc6 in HR-mediated repair (Tapia-Alveal and O'Connell, 2011). Furthermore, this complex has also been shown to play a role at stably stalled replication forks, where it is required for the recruitment of Rad52, the HR initiating protein, onto chromatin without apparent recruitment of Rad51 .…”

Section: Introductionmentioning

confidence: 99%

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Brc1-dependent recovery from replication stress

Bass

Murray

O’Connell

2012

Journal of Cell Science

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Summary 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 brc1D 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 brc1D cells, and brc1D 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 cH2A. 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. IntroductionDuring DNA replication, cells are extremely vulnerable to DNA damage. This vulnerability is due to both the genomic lesions themselves, but also to the impediment these lesions cause to the completion of replication. Replication forks that encounter sites of DNA damage stall in the face of these lesions, a process that activates the DNA replication checkpoint to help maintain stalled fork stability, halt the cell cycle, and initiate DNA repair. The majority of replication forks are presumed to retain the full complement of replication machinery in a stable conformation at sites of fork stalling until the impeding lesion is repaired. However, some lesions are not readily resolved and so alternative mechanisms for the completion of replication have evolved. These include the collapse of the stalled replication fork and its repair through homologous recombination (HR), bypass of the DNA lesion through the post-replication repair (PRR) pathway, which includes both error-prone and error-free mechanisms, and the completion of replication through the firing of adjacent origins (Branzei and Foiani, 2008;Branzei and Foiani, 2009).PRR mediates lesion bypass through a two-step process. Initia...

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Brc1-dependent recovery from replication stress

Bass

Murray

O’Connell

2012

Journal of Cell Science

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“…In the case of the Smc5/6 mutants, this suppresses their DNA repair defects and damage sensitivity, with lesions channeled into the postreplication repair (PRR) pathway. However, in cycling cells, these vRING domain mutants do not affect the localization of the Smc5/6 complex on chromatin (17). Thus, Smc5/6 recruitment to lesions is Nse1 dependent, and it is the recruitment of mutant complexes that confers the DNA damage sensitivity.…”

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confidence: 92%

“…Apart from the SMC subunits, what distinguishes Smc5/6 from cohesin complexes are additional types of non-SMC subunits. These include a SUMO ligase (Nse2) that in S. pombe is important to survive replication stress (16,17). Further, Smc5/6 contains a vRING domain protein (Nse1) that in S. pombe is required for Smc5/6 to associate with sites of DNA damage (17) and has been suggested to have ubiquitin ligase activity (18).…”

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H2A.Z-Dependent Regulation of Cohesin Dynamics on Chromosome Arms

Tapia-Alveal

Lin

Yeoh

et al. 2014

Molecular and Cellular Biology

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Structural maintenance of chromosomes (SMC) complexes and DNA topoisomerases are major determinants of chromosome structure and dynamics. The cohesin complex embraces sister chromatids throughout interphase, but during mitosis most cohesin is stripped from chromosome arms by early prophase, while the remaining cohesin at kinetochores is cleaved at anaphase. This two-step removal of cohesin is required for sister chromatids to separate. The cohesin-related Smc5/6 complex has been studied mostly as a determinant of DNA repair via homologous recombination. However, chromosome segregation fails in Smc5/6 null mutants or cells treated with small interfering RNAs. This also occurs in Smc5/6 hypomorphs in the fission yeast Schizosaccharomyces pombe following genotoxic and replication stress, or topoisomerase II dysfunction, and these mitotic defects are due to the postanaphase retention of cohesin on chromosome arms. Here we show that mitotic and repair roles for Smc5/6 are genetically separable in S. pombe. Further, we identified the histone variant H2A.Z as a critical factor to modulate cohesin dynamics, and cells lacking H2A.Z suppress the mitotic defects conferred by Smc5/6 dysfunction. Together, H2A.Z and the SMC complexes ensure genome integrity through accurate chromosome segregation. Chromosomal integrity is essential for normal growth and development. In eukaryotes, there are three essential complexes of proteins that are central to chromosome dynamics. These are cohesin, condensin, and the Smc5/6 complex, known collectively as the structural maintenance of chromosomes (SMC) complex. Each complex shares a related architecture, and central to them are a heterodimer of SMC proteins: Smc1 and -3 in cohesin, Smc2 and -4 in condensin, and Smc5 and -6 in Smc5/6. These SMC proteins are large coiled-coil molecules with globular N and C termini containing Walker A and B ATP-binding motifs. They fold and interact at a flexible hinge, with ATP acting to hold the globular domains together. A kleisin protein bridges each heterodimer to form a putative ring-shaped structure, and each complex has specific additional non-SMC proteins that serve as regulators and effectors of function (1-3).Chromosome condensation and sister chromatid cohesion are the key roles for condensin and cohesin, respectively (1). However, defects in DNA repair have also been described for yeast strains harboring hypomorphic mutant alleles of condensin (4) and cohesin (5-7) subunits. In the case of cohesin, a role in DNA repair could stem from the fact that DNA repair by homologous recombination (HR) requires the sister chromatid to be in close proximity to the damaged chromatid. However, more recently cohesin in mammalian cells has been shown to also act as a transcriptional insulator in collaboration with CTCF (8, 9), and so the DNA repair function of cohesin may be more complex than a simple scaffolding mechanism. As its name suggests, deciphering Smc5/6 function has proved more elusive, though most studies have focused on a role for this complex in...

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Interaction of the Saccharomyces cerevisiae RING-domain protein Nse1 with Nse3 and the Smc5/6 complex is required for chromosome replication and stability

et al. 2017

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Genomic stability is maintained by the concerted actions of numerous protein complexes that participate in chromosomal duplication, repair, and segregation. The Smc5/6 complex is an essential multi-subunit complex crucial for repair of DNA double-strand breaks. Two of its subunits, Nse1 and Nse3, are homologous to the RING-MAGE complexes recently described in human cells. We investigated the contribution of the budding yeast Nse1 RING-domain by isolating a mutant nse1-103 bearing substitutions in conserved Zinc-coordinating residues of the RING-domain that is hypersensitive to genotoxic stress and temperature. The nse1-103 mutant protein was defective in interaction with Nse3 and other Smc5/6 complex subunits, Nse4 and Smc5. Chromosome loss was enhanced, accompanied by a delay in the completion of replication and a modest defect in sister chromatid cohesion, in nse1-103. The nse1-103 mutant was synthetic sick with rrm3∆ (defective in fork passage through pause sites), this defect was rescued by inactivation of Tof1, a subunit of the fork protection complex that enforces pausing. The temperature sensitivity of nse1-103 was partially suppressed by deletion of MPH1, encoding a DNA-helicase. Homology modeling of the structure of the budding yeast Nse1-Nse3 heterodimer based on the human Nse1-MAGEG1 structure suggests a similar organization and indicates that perturbation of the Zn-coordinating cluster has the potential to allosterically alter structural elements at the Nse1/Nse3 interaction interface that may abrogate their association. Our findings demonstrate that the budding yeast Nse1 RING-domain organization is important for interaction with Nse3, which is crucial for completion of chromosomal replication, cohesion, and maintenance of chromosome stability.

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Nse1-dependent recruitment of Smc5/6 to lesion-containing loci contributes to the repair defects of mutant complexes

Abstract: The Smc5/6 complex is widely believed to be required for homologous recombination. It is shown that repair defects of Smc5/6 mutants are due to the Nse1-dependent recruitment of dysfunctional complexes to lesions.

Cited by 19 publications

References 73 publications

Brc1-dependent recovery from replication stress

Brc1-dependent recovery from replication stress

H2A.Z-Dependent Regulation of Cohesin Dynamics on Chromosome Arms

Interaction of the Saccharomyces cerevisiae RING-domain protein Nse1 with Nse3 and the Smc5/6 complex is required for chromosome replication and stability

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