Interacting proteins Rtt109 and Vps75 affect the efficiency of non-homologous end-joining in Saccharomyces cerevisiae

Jessulat, Matthew; Alamgir,; Salsali, Hamid; Greenblatt, Jack; Xu, Jianhua; Golshani, Ashkan

doi:10.1016/j.abb.2007.11.001

Cited by 45 publications

(62 citation statements)

References 43 publications

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“…To identify the genes related to the NHEJ pathway, we used an established in vitro plasmid-based DSB repair assay (12) in which the linearized p416 plasmid containing the URA3 selection marker was introduced into a yeast array of target single-gene deletion mutants. The plasmid cleavage site (i.e., an induced DSB) was contained in a region with no homologous sequence available in the genome to repair (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The efficiencies of chromosomal DSBs in yeast for the selected set of strains derived from JKM139 were assayed essentially as described elsewhere (12). Briefly, overnight cultures of wild-type or mutant strains were grown in fresh YPD or YP-raffinose (1% yeast extract, 2% Bacto peptone, 2% raffinose) at 30°C to an optical density at 600 nm (OD 600 ) of ϳ0.5.…”

Section: Methodsmentioning

confidence: 99%

“…The plasmid end-joining or repair assay for NHEJ in yeast was performed essentially as previously described (12). Colony formation for linear/circular transformations for mutant replicates over linear/circular for wild-type replicates handled on the same day were used to estimate NHEJ repair efficiency.…”

Section: Methodsmentioning

confidence: 99%

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Spindle Checkpoint Factors Bub1 and Bub2 Promote DNA Double-Strand Break Repair by Nonhomologous End Joining

Jessulat

Malty

Nguyen-Tran

et al. 2015

Molecular and Cellular Biology

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The nonhomologous end-joining (NHEJ) pathway is essential for the preservation of genome integrity, as it efficiently repairs DNA double-strand breaks (DSBs). Previous biochemical and genetic investigations have indicated that, despite the importance of this pathway, the entire complement of genes regulating NHEJ remains unknown. To address this, we employed a plasmidbased NHEJ DNA repair screen in budding yeast (Saccharomyces cerevisiae) using 369 putative nonessential DNA repair-related components as queries. Among the newly identified genes associated with NHEJ deficiency upon disruption are two spindle assembly checkpoint kinases, Bub1 and Bub2. Both observation of resulting phenotypes and chromatin immunoprecipitation demonstrated that Bub1 and -2, either alone or in combination with cell cycle regulators, are recruited near the DSB, where phosphorylated Rad53 or H2A accumulates. Large-scale proteomic analysis of Bub kinases phosphorylated in response to DNA damage identified previously unknown kinase substrates on Tel1 S/T-Q sites. Moreover, Bub1 NHEJ function appears to be conserved in mammalian cells. 53BP1, which influences DSB repair by NHEJ, colocalizes with human BUB1 and is recruited to the break sites. Thus, while Bub is not a core component of NHEJ machinery, our data support its dual role in mitotic exit and promotion of NHEJ repair in yeast and mammals.T he repair of DNA double-strand breaks (DSBs) is an essential process required for the preservation of genome integrity and the normal functioning of the cell (1). These cytotoxic lesions are repaired by major DSB repair pathways, including the homologous recombination (HR) (2) and nonhomologous end-joining (NHEJ) systems (1). While the former is the prevalent pathway in the unicellular budding yeast Saccharomyces cerevisiae (3), the latter is more prevalent in mammalian cells, especially those that are quiescent (4), and can repair DNA lesions even if there is no homologous strand (5). Notably, the impairment of NHEJ in mammalian cells is frequently linked to genomic instability, cancer, and lymphoid V(D)J (i.e., variable, diversity, and joining gene segments) recombination defects. Therefore, a detailed molecular understanding of this pathway would provide critical insight into the genetic risk factors related to carcinogenesis or immunological disorders (6).As in mammalian cells, the core components of the classical NHEJ pathway in S. cerevisiae depends on three major complexes, YKu (Ku), MRX, and DNL4, which are rapidly recruited to DSBs (7). Initially, the yeast Ku heterodimer (Ku70/80) binds to each end of a DSB, serving as an anchor for protein complexes involved in securing and annealing the break, also suppressing the competing HR pathway (8). After this, the DSB processing complex MRX (Mre11-Rad50-Xrs2), which acts at an early stage of both the NHEJ and HR repair pathways (9), spans the lesions so that the DNA ligase complex, DNL4 (i.e., Dnl4-Lif1-Nej1), can rejoin the DSB ends (10).While the actions of these core protein complexes in y...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Spindle Checkpoint Factors Bub1 and Bub2 Promote DNA Double-Strand Break Repair by Nonhomologous End Joining

Jessulat

Malty

Nguyen-Tran

et al. 2015

Molecular and Cellular Biology

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“…Recent reports also indicate that modulation of H3 K56 acetylation can lead to defects in silencing (Hyland et al 2005;Xu et al 2007), suggesting that ASF1-dependent silencing phenotypes may be related to acetylation of K56 on histone H3 in addition to acetylation of K16 on histone H4. In addition, work from several groups has led to the identification of a pathway involving H3 K56 acetylation, RTT109, and ASF1 in maintaining genome integrity in response to DNA damage (Masumoto et al 2005;Ozdemir et al 2005;Celic et al 2006;Maas et al 2006;Recht et al 2006;Schneider et al 2006;Agez et al 2007;Driscoll et al 2007;Han et al 2007a,b,c;Tsubota et al 2007;Jessulat et al 2008). The findings that (1) acetylation of K56 on histone H3 affects DNA damage sensitivity and silencing, (2) a DNA-damaging agent prevents silent chromatin formation (Kirchmaier and Rine 2006;see also Miller and Nasmyth 1984;Fox et al 1997), and (3) checkpoint activation in response to DNA damage alters Sir localization and silencing (Martin et al 1999;McAinsh et al 1999;Mills et al 1999;Sharp et al 2005) have motivated us to explore the relationship between histone modification, silencing, DNA replication, and responses to DNA damage.…”

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confidence: 99%

Proliferating Cell Nuclear Antigen and ASF1 Modulate Silent Chromatin inSaccharomyces cerevisiaevia Lysine 56 on Histone H3

et al. 2008

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The formation and stability of epigenetically regulated chromatin is influenced by DNA replication and factors that modulate post-translational modifications on histones. Here we describe evidence that PCNA can affect silencing in Saccharomyces cerevisiae by facilitating deposition of H3 K56ac onto chromosomes. We propose that PCNA participates in this process through a pathway that includes replication factor C, the chromatin assembly factor Asf1p, and the K56-specific acetyltransferase Rtt109p. We show that mutation of POL30 or loss of K56-acetylation in rtt109 and histone H3 mutants enhances silencing at the crippled HMR locus HMRae** via restoring Sir binding and that pol30 mutants with silencing phenotypes have reduced levels of H3 K56ac. Although loss of acetylation on H3 K56 was generally compatible with silencing, mutations at this residue also led to defects in silencing an ADE2 reporter at HMR and abolished silencing when combined with cac1 or pol30-8. These silencing phenotypes are analogous to those in asf1 mutants or pol30-6 and pol30-79 mutants with defects in ASF1-dependent pathways. On the basis of these findings, we propose that mutations in DNA replication factors alter acetylation of H3 K56. We show that this defect, in turn, contributes to misregulation of epigenetic processes as well as of cellular responses to DNA damage.

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“…Calculation of genetic distance and genetic congruence. Growth-based genetic interactions were measured using a modified BioGRID database derived from version 2.0.60 (51), including the interaction categories "synthetic lethality," "synthetic growth defect," and "haploinsufficiency," as well as "phenotypic enhancement" data specifically derived from E-MAP studies (52)(53)(54)(55). We also added 8,102 and 191,890 E-MAP interactions from additional studies published during the course of this analysis (56,57).…”

Section: Methodsmentioning

confidence: 99%

Bioinformatic identification of genes suppressing genome instability

Putnam¹,

Allen-Soltero

Martı́nez³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Unbiased forward genetic screens for mutations causing increased gross chromosomal rearrangement (GCR) rates in Saccharomyces cerevisiae are hampered by the difficulty in reliably using qualitative GCR assays to detect mutants with small but significantly increased GCR rates. We therefore developed a bioinformatic procedure using genome-wide functional genomics screens to identify and prioritize candidate GCR-suppressing genes on the basis of the shared drug sensitivity suppression and similar genetic interactions as known GCR suppressors. The number of known suppressors was increased from 75 to 110 by testing 87 predicted genes, which identified unanticipated pathways in this process. This analysis explicitly dealt with the lack of concordance among high-throughput datasets to increase the reliability of phenotypic predictions. Additionally, shared phenotypes in one assay were imperfect predictors for shared phenotypes in other assays, indicating that although genome-wide datasets can be useful in aggregate, caution and validation methods are required when deciphering biological functions via surrogate measures, including growth-based genetic interactions.

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Interacting proteins Rtt109 and Vps75 affect the efficiency of non-homologous end-joining in Saccharomyces cerevisiae

Cited by 45 publications

References 43 publications

Spindle Checkpoint Factors Bub1 and Bub2 Promote DNA Double-Strand Break Repair by Nonhomologous End Joining

Spindle Checkpoint Factors Bub1 and Bub2 Promote DNA Double-Strand Break Repair by Nonhomologous End Joining

Proliferating Cell Nuclear Antigen and ASF1 Modulate Silent Chromatin inSaccharomyces cerevisiaevia Lysine 56 on Histone H3

Bioinformatic identification of genes suppressing genome instability

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