Zinc finger protein E4F1 cooperates with PARP-1 and BRG1 to promote DNA double-strand break repair

Zinc finger (ZnF) proteins represent one of the largest families of human proteins, although most remain uncharacterized. Given that numerous ZnF proteins are able to interact with DNA and poly(ADP ribose), there is growing interest in understanding their mechanism of action in the maintenance of genome integrity. We now report that the ZnF protein E4F transcription factor 1 (E4F1) is an actor in DNA repair. Indeed, E4F1 is rapidly recruited, in a poly(ADP ribose) polymerase (PARP)-dependent manner, to DNA breaks and promotes ATR/CHK1 signaling, DNA-end resection, and subsequent homologous recombination. Moreover, we identify E4F1 as a regulator of the ATP-dependent chromatin remodeling SWI/SNF complex in DNA repair. E4F1 binds to the catalytic subunit BRG1/SMARCA4 and together with PARP-1 mediates its recruitment to DNA lesions. We also report that a proportion of human breast cancers show amplification and overexpression of E4F1 or BRG1 that are mutually exclusive with BRCA1/2 alterations. Together, these results reveal a function of E4F1 in the DNA damage response that orchestrates proper signaling and repair of double-strand breaks and document a molecular mechanism for its essential role in maintaining genome integrity and cell survival.

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ZNF281 is recruited on DNA breaks to facilitate DNA repair by non-homologous end joining

Oncogene ◽

10.1038/s41388-019-1028-7 ◽

2019 ◽

Vol 39 (4) ◽

pp. 754-766 ◽

Cited By ~ 7

Author(s):

Sara Nicolai ◽

Robert Mahen ◽

Giuseppe Raschellà ◽

Alberto Marini ◽

Marco Pieraccioli ◽

...

Keyword(s):

Dna Damage ◽

Zinc Finger ◽

Zinc Finger Protein ◽

Dna Lesions ◽

Repair Pathway ◽

Dna Double Strand Breaks ◽

Dna Breaks ◽

End Joining ◽

Nhej Repair ◽

Non Homologous End Joining

Abstract Efficient repair of DNA double-strand breaks (DSBs) is of critical importance for cell survival. Although non-homologous end joining (NHEJ) is the most used DSBs repair pathway in the cells, how NHEJ factors are sequentially recruited to damaged chromatin remains unclear. Here, we identify a novel role for the zinc-finger protein ZNF281 in participating in the ordered recruitment of the NHEJ repair factor XRCC4 at damage sites. ZNF281 is recruited to DNA lesions within seconds after DNA damage through a mechanism dependent on its DNA binding domain and, at least in part, on poly-ADP ribose polymerase (PARP) activity. ZNF281 binds XRCC4 through its zinc-finger domain and facilitates its recruitment to damaged sites. Consequently, depletion of ZNF281 impairs the efficiency of the NHEJ repair pathway and decreases cell viability upon DNA damage. Survival analyses from datasets of commonly occurring human cancers show that higher levels of ZNF281 correlate with poor prognosis of patients treated with DNA-damaging therapies. Thus, our results define a late ZNF281-dependent regulatory step of NHEJ complex assembly at DNA lesions and suggest additional possibilities for cancer patients’ stratification and for the development of personalised therapeutic strategies.

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

Nature Communications ◽

10.1038/s41467-021-25790-2 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Sonia Jimeno ◽

Rosario Prados-Carvajal ◽

María Jesús Fernández-Ávila ◽

Sonia Silva ◽

Domenico Alessandro Silvestris ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Rna Editing ◽

Genomic Stability ◽

Strand Break ◽

Dna Lesions ◽

Dna Breaks ◽

Dna Double Strand Break ◽

Editing Enzyme

AbstractThe 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.

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Single-Strand DNA Breaks Cause Replisome Disassembly

10.1101/2020.08.17.254235 ◽

2020 ◽

Author(s):

Kyle B. Vrtis ◽

James M. Dewar ◽

Gheorghe Chistol ◽

R. Alex Wu ◽

Thomas G. W. Graham ◽

...

Keyword(s):

Mammalian Cells ◽

Genome Stability ◽

Strand Break ◽

Dna Lesions ◽

Single Strand ◽

List Type ◽

Dependent Manner ◽

Dna Breaks ◽

Leading Strand ◽

Lagging Strand

SummaryDNA damage impedes replication fork progression and threatens genome stability. Upon encounter with most DNA adducts, the replicative CMG helicase (CDC45-MCM2-7-GINS) stalls or uncouples from the point of synthesis, yet CMG eventually resumes replication. However, little is known about the effect on replication of single-strand breaks or “nicks”, which are abundant in mammalian cells. Using Xenopus egg extracts, we reveal that CMG collision with a nick in the leading strand template generates a blunt-ended double-strand break (DSB). Moreover, CMG, which encircles the leading strand template, “runs off” the end of the DSB. In contrast, CMG collision with a lagging strand nick generates a broken end with a single-stranded overhang. In this setting, CMG translocates beyond the nick on double-stranded DNA and is then actively removed from chromatin by the p97 ATPase. Our results show that nicks are uniquely dangerous DNA lesions that invariably cause replisome disassembly, and they argue that CMG cannot be deposited on dsDNA while cells resolve replication stress.HighlightsThe structures of leading and lagging strand collapsed forks are differentCMG passively “runs off” the broken DNA end during leading strand fork collapseCMG is unloaded from duplex DNA after lag collapse in a p97-dependent mannerNicks are uniquely toxic lesions that cause fork collapse and replisome disassembly

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Physical and Functional Interaction between DNA Ligase IIIα and Poly(ADP-Ribose) Polymerase 1 in DNA Single-Strand Break Repair

Molecular and Cellular Biology ◽

10.1128/mcb.23.16.5919-5927.2003 ◽

2003 ◽

Vol 23 (16) ◽

pp. 5919-5927 ◽

Cited By ~ 142

Author(s):

John B. Leppard ◽

Zhiwan Dong ◽

Zachary B. Mackey ◽

Alan E. Tomkinson

Keyword(s):

Dna Repair ◽

Zinc Finger ◽

Strand Break ◽

Single Strand ◽

Dna Ligase ◽

Strand Breaks ◽

Single Strand Breaks ◽

Parp 1 ◽

Dna Ligase Iii

ABSTRACT The repair of DNA single-strand breaks in mammalian cells is mediated by poly(ADP-ribose) polymerase 1 (PARP-1), DNA ligase IIIα, and XRCC1. Since these proteins are not found in lower eukaryotes, this DNA repair pathway plays a unique role in maintaining genome stability in more complex organisms. XRCC1 not only forms a stable complex with DNA ligase IIIα but also interacts with several other DNA repair factors. Here we have used affinity chromatography to identify proteins that associate with DNA ligase III. PARP-1 binds directly to an N-terminal region of DNA ligase III immediately adjacent to its zinc finger. In further studies, we have shown that DNA ligase III also binds directly to poly(ADP-ribose) and preferentially associates with poly(ADP-ribosyl)ated PARP-1 in vitro and in vivo. Our biochemical studies have revealed that the zinc finger of DNA ligase III increases DNA joining in the presence of either poly(ADP-ribosyl)ated PARP-1 or poly(ADP-ribose). This provides a mechanism for the recruitment of the DNA ligase IIIα-XRCC1 complex to in vivo DNA single-strand breaks and suggests that the zinc finger of DNA ligase III enables this complex and associated repair factors to locate the strand break in the presence of the negatively charged poly(ADP-ribose) polymer.

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Nizp1, a Novel Multitype Zinc Finger Protein That Interacts with the NSD1 Histone Lysine Methyltransferase through a Unique C2HR Motif

Molecular and Cellular Biology ◽

10.1128/mcb.24.12.5184-5196.2004 ◽

2004 ◽

Vol 24 (12) ◽

pp. 5184-5196 ◽

Cited By ~ 35

Author(s):

Anders Lade Nielsen ◽

Poul Jørgensen ◽

Thierry Lerouge ◽

Margarita Cerviño ◽

Pierre Chambon ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Zinc Finger ◽

Zinc Finger Protein ◽

Sotos Syndrome ◽

Dependent Manner ◽

Interacting Protein ◽

Protein Protein Interaction ◽

Histone Lysine ◽

Histone Lysine Methyltransferase ◽

Lysine Methyltransferase

ABSTRACT Haploinsufficiency of the NSD1 gene is a hallmark of Sotos syndrome, and rearrangements of this gene by translocation can cause acute myeloid leukemia. The NSD1 gene product is a SET-domain histone lysine methyltransferase that has previously been shown to interact with nuclear receptors. We describe here a novel NSD1-interacting protein, Nizp1, that contains a SCAN box, a KRAB-A domain, and four consensus C2H2-type zinc fingers preceded by a unique finger derivative, referred to herein as the C2HR motif. The C2HR motif functions to mediate protein-protein interaction with the cysteine-rich (C5HCH) domain of NSD1 in a Zn(II)-dependent fashion, and when tethered to RNA polymerase II promoters, represses transcription in an NSD1-dependent manner. Mutations of the cysteine or histidine residues in the C2HR motif abolish the interaction of Nizp1 with NSD1 and compromise the ability of Nizp1 to repress transcription. Interestingly, converting the C2HR motif into a canonical C2H2 zinc finger has a similar effect. Thus, Nizp1 contains a novel type of zinc finger motif that functions as a docking site for NSD1 and is more than just a degenerate evolutionary remnant of a C2H2 motif.

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PARP-1 protects against colorectal tumor induction, but promotes inflammation-driven colorectal tumor progression

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1712345115 ◽

2018 ◽

Vol 115 (17) ◽

pp. E4061-E4070 ◽

Cited By ~ 22

Author(s):

Bastian Dörsam ◽

Nina Seiwert ◽

Sebastian Foersch ◽

Svenja Stroh ◽

Georg Nagel ◽

...

Keyword(s):

Cell Death ◽

Dna Repair ◽

Tumor Progression ◽

Dna Methyltransferase ◽

Intestinal Inflammation ◽

Colorectal Tumor ◽

Colorectal Carcinogenesis ◽

Dependent Manner ◽

Parp 1

Colorectal cancer (CRC) is one of the most common tumor entities, which is causally linked to DNA repair defects and inflammatory bowel disease (IBD). Here, we studied the role of the DNA repair protein poly(ADP-ribose) polymerase-1 (PARP-1) in CRC. Tissue microarray analysis revealed PARP-1 overexpression in human CRC, correlating with disease progression. To elucidate its function in CRC, PARP-1 deficient (PARP-1−/−) and wild-type animals (WT) were subjected to azoxymethane (AOM)/ dextran sodium sulfate (DSS)-induced colorectal carcinogenesis. Miniendoscopy showed significantly more tumors in WT than in PARP-1−/− mice. Although the lack of PARP-1 moderately increased DNA damage, both genotypes exhibited comparable levels of AOM-induced autophagy and cell death. Interestingly, miniendoscopy revealed a higher AOM/DSS-triggered intestinal inflammation in WT animals, which was associated with increased levels of innate immune cells and proinflammatory cytokines. Tumors in WT animals were more aggressive, showing higher levels of STAT3 activation and cyclin D1 up-regulation. PARP-1−/− animals were then crossed with O6-methylguanine-DNA methyltransferase (MGMT)-deficient animals hypersensitive to AOM. Intriguingly, PARP-1−/−/MGMT−/− double knockout (DKO) mice developed more, but much smaller tumors than MGMT−/− animals. In contrast to MGMT-deficient mice, DKO animals showed strongly reduced AOM-dependent colonic cell death despite similar O6-methylguanine levels. Studies with PARP-1−/− cells provided evidence for increased alkylation-induced DNA strand break formation when MGMT was inhibited, suggesting a role of PARP-1 in the response to O6-methylguanine adducts. Our findings reveal PARP-1 as a double-edged sword in colorectal carcinogenesis, which suppresses tumor initiation following DNA alkylation in a MGMT-dependent manner, but promotes inflammation-driven tumor progression.

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Increased Sensitivity of PBMCs Isolated from Patients with Rheumatoid Arthritis to DNA Damaging Agents Is Connected with Inefficient DNA Repair

Journal of Clinical Medicine ◽

10.3390/jcm9040988 ◽

2020 ◽

Vol 9 (4) ◽

pp. 988

Author(s):

Grzegorz Galita ◽

Olga Brzezińska ◽

Izabela Gulbas ◽

Joanna Sarnik ◽

Marta Poplawska ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Dna Repair ◽

Mononuclear Cells ◽

Excision Repair ◽

Dna Lesions ◽

Dna Breaks ◽

Peripheral Blood Mononuclear ◽

Dna Damaging Agents ◽

Repair Efficiency ◽

Systemic Inflammatory Disease

Rheumatoid arthritis (RA) is a systemic, inflammatory disease of the joints and surrounding tissues. RA manifests itself with severe joint pain, articular inflammation, and oxidative stress. RA is associated with certain types of cancer. We have assumed that RA patients’ increased susceptibility to cancer may be linked with genomic instability induced by impaired DNA repair and sensitivity to DNA damaging agents. The aim of this work was to analyze the sensitivity of peripheral blood mononuclear cells (PBMCs) isolated from RA patients to DNA damaging agents: tert-butyl hydroperoxide (TBH), bleomycin, ultraviolet (UV) radiation, and methyl methanesulfonate (MMS) and calculate the repair efficiency. TBH induce oxidative DNA lesions repaired mainly by base excision repair (BER). Bleomycin induced mainly DNA double-strand breaks repaired by non-homologous end joining (NHEJ) and homologous recombination repair (HRR). We included 20 rheumatoid arthritis patients and 20 healthy controls and used an alkaline version of the comet assay with modification to measure sensitivity to DNA damaging agents and DNA repair efficiency. We found an increased number of DNA breaks and alkali-labile sites in the RA patients compared to those in the controls. Exposure to DNA damaging agents evoked the same increased damage in both groups, but we observed statistically higher PMBC sensitivity to TBH, MMS, bleomycin as well as UV. Examination of the repair kinetics of both groups revealed that the DNA lesions induced by TBH and bleomycin were more efficiently repaired in the controls than in the patients. These data suggest impaired DNA repair in RA patients, which may accelerate PBMC aging and/or lead to higher cancer incidence among RA patients.

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141 INDICATIONS FOR DNA DOUBLE-STRAND BREAK REPAIR IN EARLY BOVINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab141 ◽

2007 ◽

Vol 19 (1) ◽

pp. 188

Author(s):

A. Brero ◽

D. Koehler ◽

T. Cremer ◽

E. Wolf ◽

V. Zakhartchenko

Keyword(s):

Dna Repair ◽

Homologous Recombination ◽

Strand Break ◽

Dna Lesions ◽

Homologous Recombination Repair ◽

Dna Double Strand Breaks ◽

End Joining ◽

Male And Female ◽

Γh2ax Foci ◽

Recombination Repair

DNA double-strand breaks (DSBs) are considered the most severe type of DNA lesions, because such lesions, if unrepaired, lead to a loss of genome integrity. Soon after induction of DSBs, chromatin surrounding the damage is modified by phosphorylation of the histone variant H2AX, generating so-called γH2AX, which is a hallmark of DSBs (Takahashi et al. 2005 Cancer Lett. 229, 171–179). γH2AX appears to be a signal for the recruitment of proteins constituting the DNA repair machinery. Depending on the type of damage and the cell cycle stage of the affected cell, DSBs are repaired either by nonhomologous end joining or by homologous recombination using the sister chromatid DNA as template (Hoeijmakers 2001 Nature 411, 366–374). We used immunofluorescence to analyze chromatin composition during bovine development and found γH2AX foci in both male and female pronuclei of IVF embryos. The number and size of foci varied considerably between embryos and between the male and female pronuclei. To test whether the observed γH2AX foci represented sites of active DNA repair, we co-stained IVF zygotes for γH2AX and 3 different proteins involved in homologous recombination repair of DSBs: NBS1 (phosphorylated at amino acid serine 343), 53BP1, and Rad51. We found co-localization of γH2AX foci with phosphorylated NBS1 as well as with Rad51 but did not observe the presence of 53BP1 at γH2AX foci in IVF zygotes. Our finding shows the presence of DSBs in IVF zygotes and suggests the capability of homologous recombination repair. The lack of 53BP1, a component of homologous recombination repair, which usually co-localizes with γH2AX foci at exogenously induced DSBs (Schultz et al. 2000 J. Cell. Biol. 151, 1381–1390) poses the possibility that the mechanism present in early embryos differs substantially from that involved in DNA repair of DSBs in somatic cells.

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Mechanistic insight into the role of Poly(ADP-ribosyl)ation in DNA topology modulation and response to DNA damage

Mutagenesis ◽

10.1093/mutage/gez045 ◽

2019 ◽

Vol 35 (1) ◽

pp. 107-118

Author(s):

Bakhyt T Matkarimov ◽

Dmitry O Zharkov ◽

Murat K Saparbaev

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Genotoxic Stress ◽

Strand Break ◽

Dna Supercoiling ◽

Dna Strand Breaks ◽

Chromosomal Dna ◽

Dna Breaks ◽

Strand Breaks ◽

Dna Strand

Abstract Genotoxic stress generates single- and double-strand DNA breaks either through direct damage by reactive oxygen species or as intermediates of DNA repair. Failure to detect and repair DNA strand breaks leads to deleterious consequences such as chromosomal aberrations, genomic instability and cell death. DNA strand breaks disrupt the superhelical state of cellular DNA, which further disturbs the chromatin architecture and gene activity regulation. Proteins from the poly(ADP-ribose) polymerase (PARP) family, such as PARP1 and PARP2, use NAD+ as a substrate to catalyse the synthesis of polymeric chains consisting of ADP-ribose units covalently attached to an acceptor molecule. PARP1 and PARP2 are regarded as DNA damage sensors that, upon activation by strand breaks, poly(ADP-ribosyl)ate themselves and nuclear acceptor proteins. Noteworthy, the regularly branched structure of poly(ADP-ribose) polymer suggests that the mechanism of its synthesis may involve circular movement of PARP1 around the DNA helix, with a branching point in PAR corresponding to one complete 360° turn. We propose that PARP1 stays bound to a DNA strand break end, but rotates around the helix displaced by the growing poly(ADP-ribose) chain, and that this rotation could introduce positive supercoils into damaged chromosomal DNA. This topology modulation would enable nucleosome displacement and chromatin decondensation around the lesion site, facilitating the access of DNA repair proteins or transcription factors. PARP1-mediated DNA supercoiling can be transmitted over long distances, resulting in changes in the high-order chromatin structures. The available structures of PARP1 are consistent with the strand break-induced PAR synthesis as a driving force for PARP1 rotation around the DNA axis.

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