A Degenerate PCNA-Interacting Peptide (DPIP) box targets RNF168 to replicating DNA to limit 53BP1 signaling

The E3 ligase RNF168 has been suggested to have roles at DNA replication forks in addition to its canonical functions in DNA double-strand break (DSB) signaling. However, the precise role of RNF168 in DNA replication remains unclear. Here we demonstrate that RNF168 is recruited to DNA replication factories independent of the canonical DSB response pathway regulators and identify a degenerate PCNA-Interacting Peptide (DPIP) motif in the C-terminus of RNF168 which mediates its binding to PCNA. An RNF168 mutant harboring substitutions in the DPIP box fails to interact with PCNA and is not recruited to sites of DNA synthesis, yet fully retains its ability to promote DSB-induced 53BP1 foci. Surprisingly, the RNF168 DPIP mutant also retains the ability to support ongoing DNA replication fork movement, demonstrating that PCNA-binding is dispensable for normal S-phase functions. However, replisome-associated RNF168 functions to suppress the DSB-induced 53BP1 DNA damage response during S-phase. Moreover, we show that WT RNF168 can perform PCNA ubiquitylation independently of RAD18 and also synergizes with RAD18 to amplify PCNA ubiquitylation. Taken together, our results identify non-canonical functions of RNF168 at the replication fork and demonstrate new mechanisms of cross talk between the DNA damage and replication stress response pathways.

Download Full-text

Replication fork dynamics and the DNA damage response

Biochemical Journal ◽

10.1042/bj20112100 ◽

2012 ◽

Vol 443 (1) ◽

pp. 13-26 ◽

Cited By ~ 77

Author(s):

Rebecca M. Jones ◽

Eva Petermann

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Dna Damage Response ◽

Replication Fork ◽

Molecular Mechanisms ◽

S Phase ◽

Replication Initiation ◽

Developmental Defects ◽

Replication Fork Progression ◽

Damage Response

Prevention and repair of DNA damage is essential for maintenance of genomic stability and cell survival. DNA replication during S-phase can be a source of DNA damage if endogenous or exogenous stresses impair the progression of replication forks. It has become increasingly clear that DNA-damage-response pathways do not only respond to the presence of damaged DNA, but also modulate DNA replication dynamics to prevent DNA damage formation during S-phase. Such observations may help explain the developmental defects or cancer predisposition caused by mutations in DNA-damage-response genes. The present review focuses on molecular mechanisms by which DNA-damage-response pathways control and promote replication dynamics in vertebrate cells. In particular, DNA damage pathways contribute to proper replication by regulating replication initiation, stabilizing transiently stalled forks, promoting replication restart and facilitating fork movement on difficult-to-replicate templates. If replication fork progression fails to be rescued, this may lead to DNA damage and genomic instability via nuclease processing of aberrant fork structures or incomplete sister chromatid separation during mitosis.

Download Full-text

Succinylation at a key residue of FEN1 is involved in the DNA damage response to maintain genome stability

AJP Cell Physiology ◽

10.1152/ajpcell.00137.2020 ◽

2020 ◽

Vol 319 (4) ◽

pp. C657-C666

Author(s):

Rongyi Shi ◽

Yiyi Wang ◽

Ya Gao ◽

Xiaoli Xu ◽

Shuyu Mao ◽

...

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Replication Fork ◽

Genome Stability ◽

Damage Response ◽

Dna Replication And Repair ◽

Fork Stalling ◽

Stalled Replication Forks ◽

Maintain Genome Stability

Human flap endonuclease 1 (FEN1) is a structure-specific, multifunctional endonuclease essential for DNA replication and repair. Our previous study showed that in response to DNA damage, FEN1 interacts with the PCNA-like Rad9-Rad1-Hus1 complex instead of PCNA to engage in DNA repair activities, such as stalled DNA replication fork repair, and undergoes SUMOylation by SUMO-1. Here, we report that succinylation of FEN1 was stimulated in response to DNA replication fork-stalling agents, such as ultraviolet (UV) irradiation, hydroxyurea, camptothecin, and mitomycin C. K200 is a key succinylation site of FEN1 that is essential for gap endonuclease activity and could be suppressed by methylation and stimulated by phosphorylation to promote SUMO-1 modification. Succinylation at K200 of FEN1 promoted the interaction of FEN1 with the Rad9-Rad1-Hus1 complex to rescue stalled replication forks. Impairment of FEN1 succinylation led to the accumulation of DNA damage and heightened sensitivity to fork-stalling agents. Altogether, our findings suggest an important role of FEN1 succinylation in regulating its roles in DNA replication and repair, thus maintaining genome stability.

Download Full-text

ADAR2-mediated RNA editing of DNA:RNA hybrids is required for DNA double strand break repair

10.1101/2021.03.24.436729 ◽

2021 ◽

Author(s):

Sonia Jimeno ◽

Rosario Prados-Carvajal ◽

Maria Jesus Fernandez-Avila ◽

Sonia Silva ◽

Domenico Alessandro Silvestris ◽

...

Keyword(s):

Dna Damage ◽

Rna Editing ◽

Genomic Stability ◽

Strand Break ◽

Dna Lesions ◽

Dna Breaks ◽

Dna Double Strand Break ◽

Damage Response ◽

Editing Enzyme

The maintenance of genomic stability requires the coordination of multiple cellular tasks upon the appearance of DNA lesions. RNA editing, the post-transcriptional sequence alteration of RNA, has a profound effect on cell homeostasis, but its implication in the response to DNA damage was not previously explored. Here we show that, in response to DNA breaks, an overall change of the Adenosine-to-Inosine RNA editing is observed, a phenomenon we call the RNA Editing DAmage Response (REDAR). REDAR relies on the checkpoint kinase ATR and the recombination factor CtIP. Moreover, depletion of the RNA editing enzyme ADAR2 renders cells hypersensitive to genotoxic agents, increases genomic instability and hampers homologous recombination by impairing DNA resection. Such a role of ADAR2 in DNA repair goes beyond the recoding of specific transcripts, but depends on ADAR2 editing DNA:RNA hybrids to ease their dissolution.

Download Full-text

Repair Kinetics of Genomic Interstrand DNA Cross-Links: Evidence for DNA Double-Strand Break-Dependent Activation of the Fanconi Anemia/BRCA Pathway

Molecular and Cellular Biology ◽

10.1128/mcb.24.1.123-134.2004 ◽

2004 ◽

Vol 24 (1) ◽

pp. 123-134 ◽

Cited By ~ 170

Author(s):

Andreas Rothfuss ◽

Markus Grompe

Keyword(s):

Fanconi Anemia ◽

Strand Break ◽

S Phase ◽

Dna Double Strand Breaks ◽

Subsequent Formation ◽

Strand Breaks ◽

Dna Double Strand Break ◽

Cross Links ◽

Kinetics Of

ABSTRACT The detailed mechanisms of DNA interstrand cross-link (ICL) repair and the involvement of the Fanconi anemia (FA)/BRCA pathway in this process are not known. Present models suggest that recognition and repair of ICL in human cells occur primarily during the S phase. Here we provide evidence for a refined model in which ICLs are recognized and are rapidly incised by ERCC1/XPF independent of DNA replication. However, the incised ICLs are then processed further and DNA double-strand breaks (DSB) form exclusively in the S phase. FA cells are fully proficient in the sensing and incision of ICL as well as in the subsequent formation of DSB, suggesting a role of the FA/BRCA pathway downstream in ICL repair. In fact, activation of FANCD2 occurs slowly after ICL treatment and correlates with the appearance of DSB in the S phase. In contrast, activation is rapid after ionizing radiation, indicating that the FA/BRCA pathway is specifically activated upon DSB formation. Furthermore, the formation of FANCD2 foci is restricted to a subpopulation of cells, which can be labeled by bromodeoxyuridine incorporation. We therefore conclude that the FA/BRCA pathway, while being dispensable for the early events in ICL repair, is activated in S-phase cells after DSB have formed.

Download Full-text

Set1-dependent H3K4 methylation becomes critical for DNA replication fork progression in response to changes in S phase dynamics in Saccharomyces cerevisiae

10.1101/2020.10.26.355008 ◽

2020 ◽

Author(s):

Christophe de La Roche Saint-André ◽

Vincent Géli

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Damage ◽

Dna Replication ◽

Replication Fork ◽

Genetic Material ◽

S Phase ◽

Replication Forks ◽

H3k4 Methylation ◽

Origin Firing ◽

Phase Dynamics

AbstractDNA replication is a highly regulated process that occurs in the context of chromatin structure and is sensitive to several histone post-translational modifications. In Saccharomyces cerevisiae, the histone methylase Set1 is responsible for the transcription-dependent deposition of H3K4 methylation (H3K4me) throughout the genome. Here we show that a combination of a hypomorphic replication mutation (orc5-1) with the absence of Set1 (set1Δ) compromises the progression through S phase, and this is associated with a large increase in DNA damage. The ensuing DNA damage checkpoint activation, in addition to that of the spindle assembly checkpoint, restricts the growth of orc5-1 set1Δ. Interestingly, orc5-1 set1Δ is sensitive to the lack of RNase H activity while a reduction of histone levels is able to counterbalance the loss of Set1. We propose that the recently described Set1-dependent mitigation of transcription-replication conflicts becomes critical for growth when the replication forks accelerate due to decreased origin firing in the orc5-1 background. Furthermore, we show that an increase of reactive oxygen species (ROS) levels, likely a consequence of the elevated DNA damage, is partly responsible for the lethality in orc5-1 set1Δ.Author summaryDNA replication, that ensures the duplication of the genetic material, starts at discrete sites, termed origins, before proceeding at replication forks whose progression is carefully controlled in order to avoid conflicts with the transcription of genes. In eukaryotes, DNA replication occurs in the context of chromatin, a structure in which DNA is wrapped around proteins, called histones, that are subjected to various chemical modifications. Among them, the methylation of the lysine 4 of histone H3 (H3K4) is carried out by Set1 in Saccharomyces cerevisiae, specifically at transcribed genes. We report that, when the replication fork accelerates in response to a reduction of active origins, the absence of Set1 leads to accumulation of DNA damage. Because H3K4 methylation was recently shown to slow down replication at transcribed genes, we propose that the Set1-dependent becomes crucial to limit the occurrence of conflicts between replication and transcription caused by replication fork acceleration. In agreement with this model, stabilization of transcription-dependent structures or reduction histone levels, to limit replication fork velocity, respectively exacerbates or moderates the effect of Set1 loss. Last, but not least, we show that the oxidative stress associated to DNA damage is partly responsible for cell lethality.

Download Full-text

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

Genetics ◽

10.1093/genetics/iyab042 ◽

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.

Download Full-text

DNA Damage-Induced Phosphorylation of TRF2 Is Required for the Fast Pathway of DNA Double-Strand Break Repair

Molecular and Cellular Biology ◽

10.1128/mcb.00944-08 ◽

2009 ◽

Vol 29 (13) ◽

pp. 3597-3604 ◽

Cited By ~ 29

Author(s):

Nazmul Huda ◽

Hiromi Tanaka ◽

Marc S. Mendonca ◽

David Gilley

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Functional Role ◽

Strand Break ◽

Telomere Maintenance ◽

Telomeric Dna ◽

Dsb Repair ◽

Dna Double Strand Break ◽

Damage Response ◽

Fast Pathway

ABSTRACT Protein kinases of the phosphatidylinositol 3-kinase-like kinase family, originally known to act in maintaining genomic integrity via DNA repair pathways, have been shown to also function in telomere maintenance. Here we focus on the functional role of DNA damage-induced phosphorylation of the essential mammalian telomeric DNA binding protein TRF2, which coordinates the assembly of the proteinaceous cap to disguise the chromosome end from being recognized as a double-stand break (DSB). Previous results suggested a link between the transient induction of human TRF2 phosphorylation at threonine 188 (T188) by the ataxia telangiectasia mutated protein kinase (ATM) and the DNA damage response. Here, we report evidence that X-ray-induced phosphorylation of TRF2 at T188 plays a role in the fast pathway of DNA DSB repair. These results connect the highly transient induction of human TRF2 phosphorylation to the DNA damage response machinery. Thus, we find that a protein known to function in telomere maintenance, TRF2, also plays a functional role in DNA DSB repair.

Download Full-text

Coordinated Chromatin-Association of Fanconi Anemia Network Proteins Requires Replication-Coupled DNA Damage Recognition.

Blood ◽

10.1182/blood.v104.11.723.723 ◽

2004 ◽

Vol 104 (11) ◽

pp. 723-723

Author(s):

Alexandra Sobeck ◽

Stacie Stone ◽

Bendert deGraaf ◽

Vincenzo Costanzo ◽

Johan deWinter ◽

...

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Dna Damage Response ◽

Fanconi Anemia ◽

Bone Marrow Failure ◽

S Phase ◽

Dependent Manner ◽

Core Complex ◽

Damage Response ◽

Crosslinking Agents

Abstract Fanconi anemia (FA) is a genetic disorder characterized by hypersensitivity to DNA crosslinking agents and diverse clinical symptoms, including developmental anomalies, progressive bone marrow failure, and predisposition to leukemias and other cancers. FA is genetically heterogeneous, resulting from mutations in any of at least eleven different genes. The FA proteins function together in a pathway composed of a mulitprotein core complex that is required to trigger the DNA-damage dependent activation of the downstream FA protein, FANCD2. This activation is thought to be the key step in a DNA damage response that functionally links FA proteins to major breast cancer susceptibility proteins BRCA1 and BRCA2 (BRCA2 is FA gene FANCD1). The essential function of the FA proteins is unknown, but current models suggest that FA proteins function at the interface between cell cycle checkpoints, DNA repair and DNA replication, and are likely to play roles in the DNA damage response during S phase. To provide a platform for dissecting the key functional events during S-phase, we developed cell-free assays for FA proteins based on replicating extracts from Xenopus eggs. We identified the Xenopus homologs of human FANCD2 (xFANCD2) and several of the FA core complex proteins (xCCPs), and biochemically characterized these proteins in replicating cell-free extracts. We found that xCCPs and a modified isoform of xFANCD2 become associated with chromatin during normal and disrupted DNA replication. Blocking initiation of replication with geminin demonstrated that association of xCCPs and xFANCD2 with chromatin occurs in a strictly replication-dependent manner that is enhanced following DNA damage by crosslinking agents or by addition of aphidicolin, an inhibitor of replicative DNA polymerases. In addition, chromatin binding of xFANCD2, but not xBRCA2, is abrogated when xFANCA is quantitatively depleted from replicating extracts suggesting that xFANCA promotes the loading of xFANCD2 on chromatin. The chromatin-association of xFANCD2 and xCCPs is diminished in the presence of caffeine, an inhibitor of checkpoint kinases. Taken together, our data suggest a model in which the ordered loading of FA proteins on chromatin is required for processing a subset of DNA replication-blocking lesions that are resolved during late stages of replication.

Download Full-text

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

mBio ◽

10.1128/mbio.01192-18 ◽

2018 ◽

Vol 9 (4) ◽

Cited By ~ 5

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.

Download Full-text

Identification of S-phase DNA damage-response targets in fission yeast reveals conservation of damage-response networks

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1525620113 ◽

2016 ◽

Vol 113 (26) ◽

pp. E3676-E3685 ◽

Cited By ~ 8

Author(s):

Nicholas A. Willis ◽

Chunshui Zhou ◽

Andrew E. H. Elia ◽

Johanne M. Murray ◽

Antony M. Carr ◽

...

Keyword(s):

Gene Expression ◽

Dna Damage ◽

Dna Replication ◽

Fission Yeast ◽

Dna Damage Response ◽

Cellular Response ◽

Genome Stability ◽

Biological Significance ◽

S Phase ◽

Damage Response

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.

Download Full-text