scholarly journals Defects in DNA Lesion Bypass Lead to Spontaneous Chromosomal Rearrangements and Increased Cell Death

2009 ◽  
Vol 9 (2) ◽  
pp. 315-324 ◽  
Author(s):  
Kristina H. Schmidt ◽  
Emilie B. Viebranz ◽  
Lorena B. Harris ◽  
Hamed Mirzaei-Souderjani ◽  
Salahuddin Syed ◽  
...  
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Genetic Interaction ◽  
Chromosomal Rearrangements ◽  
Point Mutations ◽  
Normal Growth ◽  
Dna Helicase ◽  
Dna Damage Checkpoint ◽  
Lesion Bypass

ABSTRACT Rev3 polymerase and Mph1 DNA helicase participate in error-prone and error-free pathways, respectively, for the bypassing of template lesions during DNA replication. Here we have investigated the role of these pathways and their genetic interaction with recombination factors, other nonreplicative DNA helicases, and DNA damage checkpoint components in the maintenance of genome stability, viability, and sensitivity to the DNA-damaging agent methyl methanesulfonate (MMS). We find that cells lacking Rev3 and Mph1 exhibit a synergistic, Srs2-dependent increase in the rate of accumulating spontaneous, gross chromosomal rearrangements, suggesting that the suppression of point mutations by deletion of REV3 may lead to chromosomal rearrangements. While mph1Δ is epistatic to homologous recombination (HR) genes, both Rad51 and Rad52, but not Rad59, are required for normal growth of the rev3Δ mutant and are essential for survival of rev3Δ cells during exposure to MMS, indicating that Mph1 acts in a Rad51-dependent, Rad59-independent subpathway of HR-mediated lesion bypass. Deletion of MPH1 helicase leads to synergistic DNA damage sensitivity increases in cells with chl1Δ or rrm3Δ helicase mutations, whereas mph1Δ is hypostatic to sgs1Δ. Previously reported slow growth of mph1Δ srs2Δ cells is accompanied by G2/M arrest and fully suppressed by disruption of the Mec3-dependent DNA damage checkpoint. We propose a model for replication fork rescue mediated by translesion DNA synthesis and homologous recombination that integrates the role of Mph1 in unwinding D loops and its genetic interaction with Rev3 and Srs2-regulated pathways in the suppression of spontaneous genome rearrangements and in mutation avoidance.

scholarly journals Mrc1 Is Required for Sister Chromatid Cohesion To Aid in Recombination Repair of Spontaneous Damage

2004 ◽  
Vol 24 (16) ◽  
pp. 7082-7090 ◽  
Author(s):  
Hong Xu ◽  
Charles Boone ◽  
Hannah L. Klein
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Homologous Recombination ◽  
Sister Chromatid ◽  
Synthetic Lethality ◽  
Dna Helicase ◽  
Sister Chromatid Cohesion ◽  
Checkpoint Response ◽  
Chromatid Cohesion

ABSTRACT The SRS2 gene of Saccharomyces cerevisiae encoding a 3′→5′ DNA helicase is part of the postreplication repair pathway and functions to ensure proper repair of DNA damage arising during DNA replication through pathways that do not involve homologous recombination. Through a synthetic gene array analysis, genes that are essential when Srs2 is absent have been identified. Among these are MRC1, TOF1, and CSM3, which mediate the intra-S checkpoint response. srs2Δ mrc1Δ synthetic lethality is due to inappropriate recombination, as the lethality can be suppressed by genetic elimination of homologous recombination. srs2Δ mrc1Δ synthetic lethality is dependent on the role of Mrc1 in DNA replication but independent of the role of Mrc1 in a DNA damage checkpoint response. mrc1Δ, tof1Δ and csm3Δ mutants have sister chromatid cohesion defects, implicating sister chromatid cohesion established at the replication fork as an important factor in promoting repair of stalled replication forks through gap repair.

scholarly journals Saccharomyces cerevisiae Rrm3p DNA Helicase Promotes Genome Integrity by Preventing Replication Fork Stalling: Viability of rrm3 Cells Requires the Intra-S-Phase Checkpoint and Fork Restart Activities

2004 ◽  
Vol 24 (8) ◽  
pp. 3198-3212 ◽  
Author(s):  
Jorge Z. Torres ◽  
Sandra L. Schnakenberg ◽  
Virginia A. Zakian
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Replication Fork ◽  
Opportunity To Learn ◽  
Normal Growth ◽  
Dna Helicase ◽  
S Phase ◽  
Exogenous Dna ◽  
Fork Stalling ◽  
S Phase Checkpoint

ABSTRACT Rrm3p is a 5′-to-3′ DNA helicase that helps replication forks traverse protein-DNA complexes. Its absence leads to increased fork stalling and breakage at over 1,000 specific sites located throughout the Saccharomyces cerevisiae genome. To understand the mechanisms that respond to and repair rrm3-dependent lesions, we carried out a candidate gene deletion analysis to identify genes whose mutation conferred slow growth or lethality on rrm3 cells. Based on synthetic phenotypes, the intra-S-phase checkpoint, the SRS2 inhibitor of recombination, the SGS1/TOP3 replication fork restart pathway, and the MRE11/RAD50/XRS2 (MRX) complex were critical for viability of rrm3 cells. DNA damage checkpoint and homologous recombination genes were important for normal growth of rrm3 cells. However, the MUS81/MMS4 replication fork restart pathway did not affect growth of rrm3 cells. These data suggest a model in which the stalled and broken forks generated in rrm3 cells activate a checkpoint response that provides time for fork repair and restart. Stalled forks are converted by a Rad51p-mediated process to intermediates that are resolved by Sgs1p/Top3p. The rrm3 system provides a unique opportunity to learn the fate of forks whose progress is impaired by natural impediments rather than by exogenous DNA damage.

scholarly journals Schizosaccharomyces pombe KAT5 contributes to resection and repair of a DNA double strand break

Genetics ◽  
2021 ◽  
Author(s):  
Tingting Li ◽  
Ruben C Petreaca ◽  
Susan L Forsburg
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Chromatin Remodeling ◽  
Dna Damage Response ◽  
Histone Acetyltransferase ◽  
Strand Break ◽  
Double Strand Break ◽  
Dna Damage Checkpoint ◽  
Dna Double Strand Break ◽  
Damage Response

Abstract Chromatin remodeling is essential for effective repair of a DNA double strand break. KAT5 (S. pombe Mst1, human TIP60) is a MYST family histone acetyltransferase conserved from yeast to humans that coordinates various DNA damage response activities at a DNA double strand break (DSB), including histone remodeling and activation of the DNA damage checkpoint. In S. pombe, mutations in mst1+ causes sensitivity to DNA damaging drugs. Here we show that Mst1 is recruited to DSBs. Mutation of mst1+ disrupts recruitment of repair proteins and delays resection. These defects are partially rescued by deletion of pku70, which has been previously shown to antagonize repair by homologous recombination. These phenotypes of mst1 are similar to pht1-4KR, a non-acetylatable form of histone variant H2A.Z, which has been proposed to affect resection. Our data suggest that Mst1 functions to direct repair of DSBs towards homologous recombination pathways by modulating resection at the double strand break.

scholarly journals LATS1/WARTS phosphorylates MYPT1 to counteract PLK1 and regulate mammalian mitotic progression

2012 ◽  
Vol 197 (5) ◽  
pp. 625-641 ◽  
Author(s):  
Tatsuyuki Chiyoda ◽  
Naoyuki Sugiyama ◽  
Takatsune Shimizu ◽  
Hideaki Naoe ◽  
Yusuke Kobayashi ◽  
...  
Keyword(s):  
Dna Damage ◽  
Deoxyribonucleic Acid ◽  
Mitotic Exit ◽  
Dna Damage Checkpoint ◽  
Mitotic Progression ◽  
Myosin Phosphatase ◽  
G2 Checkpoint ◽  
Mitotic Exit Network ◽  
Kinase Screening

In the mitotic exit network of budding yeast, Dbf2 kinase phosphorylates and regulates Cdc14 phosphatase. In contrast, no phosphatase substrates of LATS1/WARTS kinase, the mammalian equivalent of Dbf2, has been reported. To address this discrepancy, we performed phosphoproteomic screening using LATS1 kinase. Screening identified MYPT1 (myosin phosphatase–targeting subunit 1) as a new substrate for LATS1. LATS1 directly and preferentially phosphorylated serine 445 (S445) of MYPT1. An MYPT1 mutant (S445A) failed to dephosphorylate Thr 210 of PLK1 (pololike kinase 1), thereby activating PLK1. This suggests that LATS1 promotes MYPT1 to antagonize PLK1 activity. Consistent with this, LATS1-depleted HeLa cells or fibroblasts from LATS1 knockout mice showed increased PLK1 activity. We also found deoxyribonucleic acid (DNA) damage–induced LATS1 activation caused PLK1 suppression via the phosphorylation of MYPT1 S445. Furthermore, LATS1 knockdown cells showed reduced G2 checkpoint arrest after DNA damage. These results indicate that LATS1 phosphorylates a phosphatase as does the yeast Dbf2 and demonstrate a novel role of LATS1 in controlling PLK1 at the G2 DNA damage checkpoint.

53BP1: function and mechanisms of focal recruitment

2009 ◽  
Vol 37 (4) ◽  
pp. 897-904 ◽  
Author(s):  
Jennifer E. FitzGerald ◽  
Muriel Grenon ◽  
Noel F. Lowndes
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Damage Response ◽  
Dna Damage Checkpoint ◽  
Checkpoint Activation ◽  
Damage Response ◽  
Nuclear Structures ◽  
Genotoxic Insult ◽  
Covalent Histone Modifications

53BP1 (p53-binding protein 1) is classified as a mediator/adaptor of the DNA-damage response, and is recruited to nuclear structures termed foci following genotoxic insult. In the present paper, we review the functions of 53BP1 in DNA-damage checkpoint activation and DNA repair, and the mechanisms of its recruitment and activation following DNA damage. We focus in particular on the role of covalent histone modifications in this process.

scholarly journals Regulation and pharmacological targeting of RAD51 in cancer

NAR Cancer ◽  
2020 ◽  
Vol 2 (3) ◽  
Author(s):  
McKenzie K Grundy ◽  
Ronald J Buckanovich ◽  
Kara A Bernstein
Keyword(s):  
Ovarian Cancer ◽  
Dna Damage ◽  
Homologous Recombination ◽  
Patient Outcomes ◽  
Cancer Biology ◽  
Cancer Resistance ◽  
Breast And Ovarian Cancer ◽  
Resistance To Therapy ◽  
Damage Repair

Abstract Regulation of homologous recombination (HR) is central for cancer prevention. However, too little HR can increase cancer incidence, whereas too much HR can drive cancer resistance to therapy. Importantly, therapeutics targeting HR deficiency have demonstrated a profound efficacy in the clinic improving patient outcomes, particularly for breast and ovarian cancer. RAD51 is central to DNA damage repair in the HR pathway. As such, understanding the function and regulation of RAD51 is essential for cancer biology. This review will focus on the role of RAD51 in cancer and beyond and how modulation of its function can be exploited as a cancer therapeutic.

scholarly journals The Main Role of Srs2 in DNA Repair Depends on Its Helicase Activity, Rather than on Its Interactions with PCNA or Rad51

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Alex Bronstein ◽  
Lihi Gershon ◽  
Gilad Grinberg ◽  
Elisa Alonso-Perez ◽  
Martin Kupiec
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Genome Stability ◽  
Dna Helicase ◽  
Main Role ◽  
Helicase Domain ◽  
Helicase Activity ◽  
C Terminus ◽  
Srs2 Helicase

ABSTRACTHomologous recombination (HR) is a mechanism that repairs a variety of DNA lesions. Under certain circumstances, however, HR can generate intermediates that can interfere with other cellular processes such as DNA transcription or replication. Cells have therefore developed pathways that abolish undesirable HR intermediates. TheSaccharomyces cerevisiaeyeast Srs2 helicase has a major role in one of these pathways. Srs2 also works during DNA replication and interacts with the clamp PCNA. The relative importance of Srs2’s helicase activity, Rad51 removal function, and PCNA interaction in genome stability remains unclear. We created a newSRS2allele [srs2(1-850)] that lacks the whole C terminus, containing the interaction site for Rad51 and PCNA and interactions with many other proteins. Thus, the new allele encodes an Srs2 protein bearing only the activity of the DNA helicase. We find that the interactions of Srs2 with Rad51 and PCNA are dispensable for the main role of Srs2 in the repair of DNA damage in vegetative cells and for proper completion of meiosis. On the other hand, it has been shown that in cells impaired for the DNA damage tolerance (DDT) pathways, Srs2 generates toxic intermediates that lead to DNA damage sensitivity; we show that this negative Srs2 activity requires the C terminus of Srs2. Dissection of the genetic interactions of thesrs2(1-850) allele suggest a role for Srs2’s helicase activity in sister chromatid cohesion. Our results also indicate that Srs2’s function becomes more central in diploid cells.IMPORTANCEHomologous recombination (HR) is a key mechanism that repairs damaged DNA. However, this process has to be tightly regulated; failure to regulate it can lead to genome instability. The Srs2 helicase is considered a regulator of HR; it was shown to be able to evict the recombinase Rad51 from DNA. Cells lacking Srs2 exhibit sensitivity to DNA-damaging agents, and in some cases, they display defects in DNA replication. The relative roles of the helicase and Rad51 removal activities of Srs2 in genome stability remain unclear. To address this question, we created a new Srs2 mutant which has only the DNA helicase domain. Our study shows that only the DNA helicase domain is needed to deal with DNA damage and assist in DNA replication during vegetative growth and in meiosis. Thus, our findings shift the view on the role of Srs2 in the maintenance of genome integrity.

scholarly journals Mph1p promotes gross chromosomal rearrangement through partial inhibition of homologous recombination

2008 ◽  
Vol 181 (7) ◽  
pp. 1083-1093 ◽  
Author(s):  
Soma Banerjee ◽  
Stephanie Smith ◽  
Ji-Hyun Oum ◽  
Hung-Jiun Liaw ◽  
Ji-Young Hwang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Genomic Instability ◽  
Chromosomal Rearrangement ◽  
Protein A ◽  
Replication Protein A ◽  
Dna Helicase ◽  
Double Strand Breaks ◽  
Methyl Methanesulfonate ◽  
Strand Breaks

Gross chromosomal rearrangement (GCR) is a type of genomic instability associated with many cancers. In yeast, multiple pathways cooperate to suppress GCR. In a screen for genes that promote GCR, we identified MPH1, which encodes a 3′–5′ DNA helicase. Overexpression of Mph1p in yeast results in decreased efficiency of homologous recombination (HR) as well as delayed Rad51p recruitment to double-strand breaks (DSBs), which suggests that Mph1p promotes GCR by partially suppressing HR. A function for Mph1p in suppression of HR is further supported by the observation that deletion of both mph1 and srs2 synergistically sensitize cells to methyl methanesulfonate-induced DNA damage. The GCR-promoting activity of Mph1p appears to depend on its interaction with replication protein A (RPA). Consistent with this observation, excess Mph1p stabilizes RPA at DSBs. Furthermore, spontaneous RPA foci at DSBs are destabilized by the mph1Δ mutation. Therefore, Mph1p promotes GCR formation by partially suppressing HR, likely through its interaction with RPA.

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