DNA Cross-Link Repair Protein SNM1A Interacts with PIAS1 in Nuclear Focus Formation

ABSTRACT The yeast SNM1/PSO2 gene specifically functions in DNA interstrand cross-link (ICL) repair, and its role has been suggested to be separate from other DNA repair pathways. In vertebrates, there are three homologs of SNM1 (SNM1A, SNM1B, and SNM1C/Artemis; SNM1 family proteins) whose functions are largely unknown. We disrupted each of the SNM1 family genes in the chicken B-cell line DT40. Both SNM1A- and SNM1B-deficient cells were sensitive to cisplatin but not to X-rays, whereas SNM1C/Artemis-deficient cells exhibited sensitivity to X-rays but not to cisplatin. SNM1A was nonepistatic with XRCC3 (homologous recombination), RAD18 (translesion synthesis), FANCC (Fanconi anemia), and SNM1B in ICL repair. SNM1A protein formed punctate nuclear foci depending on the conserved SNM1 (metallo-β-lactamase) domain. PIAS1 was found to physically interact with SNM1A, and they colocalized at nuclear foci. Point mutations in the SNM1 domain, which disrupted the interaction with PIAS1, led to mislocalization of SNM1A in the nucleus and loss of complementation of snm1a cells. These results suggest that interaction between SNM1A and PIAS1 is required for ICL repair.

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Mutational signatures are jointly shaped by DNA damage and repair

Nature Communications ◽

10.1038/s41467-020-15912-7 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 19

Author(s):

Nadezda V. Volkova ◽

Bettina Meier ◽

Víctor González-Huici ◽

Simone Bertolini ◽

Santiago Gonzalez ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Excision Repair ◽

Point Mutations ◽

Mutational Signatures ◽

Dna Damage And Repair ◽

C Elegans ◽

Dna Repair Deficiency ◽

Dna Alterations ◽

Repair Pathways

AbstractCells possess an armamentarium of DNA repair pathways to counter DNA damage and prevent mutation. Here we use C. elegans whole genome sequencing to systematically quantify the contributions of these factors to mutational signatures. We analyse 2,717 genomes from wild-type and 53 DNA repair defective backgrounds, exposed to 11 genotoxins, including UV-B and ionizing radiation, alkylating compounds, aristolochic acid, aflatoxin B1, and cisplatin. Combined genotoxic exposure and DNA repair deficiency alters mutation rates or signatures in 41% of experiments, revealing how different DNA alterations induced by the same genotoxin are mended by separate repair pathways. Error-prone translesion synthesis causes the majority of genotoxin-induced base substitutions, but averts larger deletions. Nucleotide excision repair prevents up to 99% of point mutations, almost uniformly across the mutation spectrum. Our data show that mutational signatures are joint products of DNA damage and repair and suggest that multiple factors underlie signatures observed in cancer genomes.

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DNA Repair Biosensor-Identified DNA Damage Activities of Endophyte Extracts from Garcinia cowa

Biomolecules ◽

10.3390/biom10121680 ◽

2020 ◽

Vol 10 (12) ◽

pp. 1680

Author(s):

Tassanee Lerksuthirat ◽

Rakkreat Wikiniyadhanee ◽

Sermsiri Chitphuk ◽

Wasana Stitchantrakul ◽

Somponnat Sampattavanich ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Fluorescent Proteins ◽

Strand Break ◽

Control Group ◽

Focus Formation ◽

Dna Damage And Repair ◽

Biological Compounds ◽

Repair Pathways ◽

Garcinia Cowa

Recent developments in chemotherapy focus on target-specific mechanisms, which occur only in cancer cells and minimize the effects on normal cells. DNA damage and repair pathways are a promising target in the treatment of cancer. In order to identify novel compounds targeting DNA repair pathways, two key proteins, 53BP1 and RAD54L, were tagged with fluorescent proteins as indicators for two major double strand break (DSB) repair pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR). The engineered biosensor cells exhibited the same DNA repair properties as the wild type. The biosensor cells were further used to investigate the DNA repair activities of natural biological compounds. An extract from Phyllosticta sp., the endophyte isolated from the medicinal plant Garcinia cowa Roxb. ex Choisy, was tested. The results showed that the crude extract induced DSB, as demonstrated by the increase in the DNA DSB marker γH2AX. The damaged DNA appeared to be repaired through NHEJ, as the 53BP1 focus formation in the treated fraction was higher than in the control group. In conclusion, DNA repair-based biosensors are useful for the preliminary screening of crude extracts and biological compounds for the identification of potential targeted therapeutic drugs.

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The dual role of the RETINOBLASTOMA-RELATED protein in the DNA damage response is spatio-temporally coordinated by the interaction with LXCXE-containing proteins.

10.1101/2021.12.22.473497 ◽

2021 ◽

Author(s):

Jorge Zamora-Zaragoza ◽

Katinka Klap ◽

Renze Heidstra ◽

Wenkun Zhou ◽

Ben Scheres

Keyword(s):

Dna Damage ◽

Cell Death ◽

Dna Repair ◽

Cell Division ◽

Dna Damage Response ◽

Growth Arrest ◽

Focus Formation ◽

Damage Response ◽

Nuclear Foci ◽

Lxcxe Motif

Living organisms face threats to genome integrity caused by environmental challenges or metabolic errors in proliferating cells. To avoid the spread of mutations, cell division is temporarily arrested while repair mechanisms deal with DNA lesions. Afterwards, cells either resume division or respond to unsuccessful repair by withdrawing from the cell cycle and undergoing cell death. How the success rate of DNA repair connects to the execution of cell death remains incompletely known, particularly in plants. Here we provide evidence that the Arabidopsis thaliana RETINOBLASTOMA-RELATED1 (RBR) protein, shown to play structural and transcriptional functions in the DNA damage response (DDR), coordinates these processes in time by successive interactions through its B-pocket sub-domain. Upon DNA damage induction, RBR forms nuclear foci; but the N849F substitution in the B-pocket, which specifically disrupts binding to LXCXE motif-containing proteins, abolishes RBR focus formation and leads to growth arrest. After RBR focus formation, the stress-responsive gene NAC044 arrests cell division. As RBR is released from nuclear foci, it can be bound by the conserved LXCXE motif in NAC044. RBR-mediated cell survival is inhibited by the interaction with NAC044. Disruption of NAC044-RBR interaction impairs the cell death response but is less important for NAC044 mediated growth arrest. Noteworthy, unlike many RBR interactors, NAC044 binds to RBR independent of RBR phosphorylation. Our findings suggest that the availability of the RBR B-pocket to interact with LXCXE-containing proteins couples the structural DNA repair functions and the transcriptional functions of RBR in the cell death program.

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FANCD2 Localizes to Cross-Linked Induced Nuclear Foci That Are Distinct From Those to Which αaII Spectrin and the Cross-Link Repair Protein, XPF, Co-Localize, Indicating An Involvement of These Proteins in Different Steps of the DNA Interstrand Cross-Link Repair Process.

Blood ◽

10.1182/blood.v114.22.3196.3196 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3196-3196

Author(s):

Pan Zhang ◽

Deepa Sridharan ◽

Michael Acosta ◽

Muriel Lambert

Keyword(s):

Time Course ◽

Repair Process ◽

Myelogenous Leukemia ◽

Cross Linking ◽

Cross Link ◽

Repair Protein ◽

Interstrand Cross Links ◽

Cross Links ◽

Nuclear Foci ◽

The Cross

Abstract Abstract 3196 Poster Board III-133 The hereditary bone marrow failure disorder, Fanconi anemia (FA), is characterized by a markedly increased incidence of acute myelogenous leukemia, diverse congenital abnormalities and a defect in ability to repair DNA interstrand cross-links. We have previously shown that in FA cells there is a deficiency in the structural protein nonerythroid a spectrin (aSpII), which is involved in repair of DNA interstrand cross-links and binds to cross-linked DNA. aSpII co-localizes in nuclear foci with FANCA and the cross-link repair protein, XPF, after normal human cells are damaged with a DNA interstrand cross-linking agent. One of the FA proteins which is thought to play an important role in the repair of DNA interstrand cross-links is FANCD2, which is known to form nuclear foci after cross-link damage. The present study was undertaken in order to get a better understanding of the relationship between aSpII and FANCD2, whether they interact with each other during the DNA repair process and co-localize in damage-induced nuclear foci. Immunofluorescence microscopy was carried out to determine whether these proteins co-localized in nuclear foci after cells were damaged with a DNA interstrand cross-linking agent, 8-methylpsoralen plus UVA light (8-MOP) or mitomycin C (MMC). Time course measurements showed that FANCD2 foci were first visible at 2 hours after damage and increased up to 16 hours and were still present at 72 hours after damage. This time course of foci formation correlated with levels of monoubiquitination of FANCD2. Measurement of gH2AX foci formation showed that the time course of foci formation was similar to that of FANCD2 measured up to 72 hours post damage. In contrast, aSpII foci were first visible between 8-10 hours after damage. The number of these foci peaked at 16 hours and by 24 hours foci were no longer observed. Co-localization studies showed that there was little co-localization of the FANCD2 and aSpII foci over this time course. This indicates that these two proteins may be involved in different steps in the DNA interstrand cross-link repair process. Based on models that have been proposed for the role of FANCD2 in the repair of DNA interstrand cross-links, we propose that, after DNA damage, FANCD2 localizes at DNA replication forks stalled at sites of interstrand cross-links and aids in the assembly of proteins at this site. This is followed by localization of aSpII and XPF and other proteins involved in the initial incision steps in DNA interstrand cross-link repair where they play a role in the unhooking of the cross-link. FANCD2 is then involved in subsequent steps in the repair process, which involve homologous recombination. Thus two proteins, FANCD2 and aSpII, both of which have been shown to be critical for the DNA interstrand cross-link repair process may be involved in different or distinct steps in this repair process. Deficiencies in these proteins would impact on DNA interstrand cross-link repair and, as we have shown for aIISp, would have an adverse effect on the genomic stability of FA cells. . Disclosures No relevant conflicts of interest to declare.

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Ubiquitin and SUMO signalling in DNA repair

Biochemical Society Transactions ◽

10.1042/bst0380116 ◽

2010 ◽

Vol 38 (1) ◽

pp. 116-131 ◽

Cited By ~ 24

Author(s):

Timothy M. Thomson ◽

Marta Guerra-Rebollo

Keyword(s):

Dna Repair ◽

Nucleotide Excision Repair ◽

Excision Repair ◽

Translesion Synthesis ◽

Strand Break ◽

Cross Link ◽

Post Translational Modifications ◽

Nucleotide Excision ◽

Specific Proteins ◽

Modified Proteins

The repair of lesions and gaps in DNA follows different pathways, each mediated by specific proteins and complexes. Post-translational modifications in many of these proteins govern their activities and interactions, ultimately determining whether a particular pathway is followed. Prominent among these modifications are the addition of phosphate or ubiquitin (and ubiquitin-like) moieties that confer new binding surfaces and conformational states on the modified proteins. The present review summarizes some of consequences of ubiquitin and ubiquitin-like modifications and interactions that regulate nucleotide excision repair, translesion synthesis, double-strand break repair and interstrand cross-link repair, with the discussion of relevant examples in each pathway.

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DNA Repair in Huntington’s Disease and Spinocerebellar Ataxias: Somatic Instability and Alternative Hypotheses

Journal of Huntington s Disease ◽

10.3233/jhd-200414 ◽

2021 ◽

Vol 10 (1) ◽

pp. 165-173 ◽

Cited By ~ 1

Author(s):

Tamara Maiuri ◽

Claudia L.K. Hung ◽

Celeste Suart ◽

Nola Begeja ◽

Carlos Barba-Bazan ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Huntington's Disease ◽

Huntington’S Disease ◽

Mismatch Repair ◽

Dna Damage Repair ◽

Cross Link ◽

Somatic Instability ◽

Damage Repair ◽

Repair Protein

The use of genome wide association studies (GWAS) in Huntington’s disease (HD) research, driven by unbiased human data analysis, has transformed the focus of new targets that could affect age at onset. While there is a significant depth of information on DNA damage repair, with many drugs and drug targets, most of this development has taken place in the context of cancer therapy. DNA damage repair in neurons does not rely on DNA replication correction mechanisms. However, there is a strong connection between DNA repair and neuronal metabolism, mediated by nucleotide salvaging and the poly ADP-ribose (PAR) response, and this connection has been implicated in other age-onset neurodegenerative diseases. Validation of leads including the mismatch repair protein MSH3, and interstrand cross-link repair protein FAN1, suggest the mechanism is driven by somatic CAG instability, which is supported by the protective effect of CAA substitutions in the CAG tract. We currently do not understand: how somatic instability is triggered; the state of DNA damage within expanding alleles in the brain; whether this damage induces mismatch repair and interstrand cross-link pathways; whether instability mediates toxicity, and how this relates to human ageing. We discuss DNA damage pathways uncovered by HD GWAS, known roles of other polyglutamine disease proteins in DNA damage repair, and a panel of hypotheses for pathogenic mechanisms.

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DOG-1 Is the Caenorhabditis elegans BRIP1/FANCJ Homologue and Functions in Interstrand Cross-Link Repair

Molecular and Cellular Biology ◽

10.1128/mcb.01641-07 ◽

2007 ◽

Vol 28 (5) ◽

pp. 1470-1479 ◽

Cited By ~ 80

Author(s):

Jillian L. Youds ◽

Louise J. Barber ◽

Jordan D. Ward ◽

Spencer J. Collis ◽

Nigel J. O'Neil ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Genetic Analysis ◽

Replication Fork ◽

Cancer Susceptibility ◽

X Rays ◽

Wild Type ◽

Cross Link ◽

Focus Formation ◽

C Elegans ◽

Functional Conservation

ABSTRACT Fanconi anemia (FA) is a cancer susceptibility syndrome characterized by defective DNA interstrand cross-link (ICL) repair. Here, we show that DOG-1 is the Caenorhabditis elegans homologue of FANCJ, a helicase mutated in FA-J patients. DOG-1 performs a conserved role in ICL repair, as dog-1 mutants are hypersensitive to ICL-inducing agents, but not to UVC irradiation or X rays. Genetic analysis indicated that dog-1 is epistatic with fcd-2 (C. elegans FANCD2) but is nonepistatic with brc-1 (C. elegans BRCA1), thus establishing the existence of two distinct pathways of ICL repair in worms. Furthermore, DOG-1 is dispensable for FCD-2 and RAD-51 focus formation, suggesting that DOG-1 operates downstream of FCD-2 and RAD-51 in ICL repair. DOG-1 was previously implicated in poly(G)/poly(C) (G/C) tract maintenance during DNA replication. G/C tracts remain stable in the absence of ATL-1, CLK-2 (FA pathway activators), FCD-2, BRC-2, and MLH-1 (associated FA components), implying that DOG-1 is the sole FA component required for G/C tract maintenance in a wild-type background. However, FCD-2 is required to promote deletion-free repair at G/C tracts in dog-1 mutants, consistent with a role for FA factors at the replication fork. The functional conservation between DOG-1 and FANCJ suggests a possible role for FANCJ in G/C tract maintenance in human cells.

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The HMCES DNA-protein cross-link functions as a constitutive DNA repair intermediate

10.1101/2021.07.29.454365 ◽

2021 ◽

Author(s):

Daniel R Semlow ◽

Victoria A MacKrell ◽

Johannes Walter

Keyword(s):

Dna Repair ◽

Strand Break ◽

S Phase ◽

Cross Link ◽

Link Functions ◽

Cross Links ◽

Ap Sites ◽

Repair Pathways ◽

Break Formation ◽

Ap Site

The HMCES protein forms a covalent DNA-protein cross-link (DPC) with abasic (AP) sites in ssDNA, and the resulting HMCES-DPC is thought to suppress double-strand break formation in S phase. However, the dynamics of HMCES cross-linking and whether any DNA repair pathways normally include an HMCES-DPC intermediate remain unknown. Here, we show that an HMCES-DPC forms efficiently on the AP site generated during replication-coupled DNA interstrand cross-link (ICL) repair. We use this system to show that HMCES cross-links form on DNA after the replicative CMG helicase has passed over the AP site, and that HMCES is subsequently removed by the SPRTN protease. The HMCES-DPC suppresses DSB formation, slows translesion synthesis (TLS) past the AP site, and introduces a bias for insertion of deoxyguanosine opposite the AP site. These data show that HMCES-DPCs can form as constitutive intermediates in replication-coupled repair, and they suggest a general model of how HMCES protects AP sites during DNA replication.

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FANCF Co-Localizes with Nonerythroid Alpha Spectrin in Nuclear Foci and Shows Enhanced Binding to It in Normal but Not Fanconi Anemia, Group A, Cells after Damage with a DNA Interstrand Cross-Linking Agent.

Blood ◽

10.1182/blood.v110.11.838.838 ◽

2007 ◽

Vol 110 (11) ◽

pp. 838-838

Author(s):

Deepa M. Sridharan ◽

Laura W. McMahon ◽

Muriel W. Lambert

Keyword(s):

Dna Repair ◽

Fanconi Anemia ◽

Repair Process ◽

Cross Linking ◽

Cross Link ◽

A Cells ◽

Interstrand Cross Links ◽

Cross Links ◽

Normal Human ◽

Nuclear Foci

Abstract Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, a predisposition to cancer, congenital abnormalities and a cellular hypersensitivity to DNA interstrand cross-linking agents, which correlates with a defect in ability to repair interstrand cross-links. We have previously shown that in FA cells there is a deficiency in the structural protein nonerythroid a spectrin (aSpII), which is involved in repair of DNA interstrand cross-links and binds to cross-linked DNA. aSpII co-localizes in damage-induced nuclear foci with FANCA and the cross-link repair protein, XPF, after normal human cells are damaged with a DNA interstrand cross-linking agent. The present study was undertaken in order to get a better understanding of the relationship between aSpII and the FA proteins and the functional importance of this relationship in the repair of DNA interstrand cross-links and the repair defect in FA cells. Immunofluorescence microscopy was carried out to determine whether, after damage, additional FA proteins co-localize with aSpII in nuclear foci and whether the interaction between these proteins is enhanced after cross-link damage. The results show that in normal human cells another FA core complex protein, FANCF, co-localizes with aSpII in nuclear foci after cells are damaged with a DNA interstrand cross-linking agent, 8-methylpsoralen plus UVA light (8-MOP). Time course measurements show that these FANCF/aSpII foci are first visible between 6–8 hours after damage and the number of these foci peaks at 16 hours. By 24 hours after exposure, foci are no longer observed. This is the same time frame previously observed for formation and co-localization of FANCA and XPF foci with aSpII. In contrast, in FA-A cells, which are not deficient in FANCF, very few damage induced FANCF or aSpII foci are observed. In corrected FA-A cells, expressing the FANCA cDNA, FANCF and aSpII again co-localize in discrete foci in the nucleus after damage. Co-localization of FANCF in damage-induced foci with aSpII correlates with enhanced binding of FANCF to aSpII after damage. Co-immunoprecipitation studies show that after normal cells are damaged with 8-MOP there is enhanced binding of FANCF, as well as FANCA, to aSpII in the damaged cells compared to this binding in undamaged cells. This further indicates that there is an important interaction between FANCF, FANCA and aSpII during the repair process. These results support our model that aSpII plays a pivotal role in the recruitment of FA and DNA repair proteins to sites of damage where it acts as a scaffold aiding in their interactions with each other or with damaged DNA, thus enhancing the DNA repair process. In FA cells, where there is a deficiency in aSpII, this recruitment is defective as are the interactions of proteins at these sites. This correlates with the reduced repair of interstrand cross-links in FA cells. Thus a deficiency in the interaction of these FA proteins with aSpII may be an important factor in the defective DNA repair pathway in FA cells.

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hMre11 and hRad50 nuclear foci are induced during the normal cellular response to DNA double-strand breaks.

Molecular and Cellular Biology ◽

10.1128/mcb.17.10.6087 ◽

1997 ◽

Vol 17 (10) ◽

pp. 6087-6096 ◽

Cited By ~ 331

Author(s):

R S Maser ◽

K J Monsen ◽

B E Nelms ◽

J H Petrini

Keyword(s):

Cellular Response ◽

Strand Break ◽

Response To Treatment ◽

Dna Double Strand Breaks ◽

Dsb Repair ◽

Focus Formation ◽

Strand Breaks ◽

Dna Double Strand Break ◽

Repair Protein ◽

Nuclear Foci

We previously identified a conserved multiprotein complex that includes hMre11 and hRad50. In this study, we used immunofluorescence to investigate the role of this complex in DNA double-strand break (DSB) repair. hMre11 and hRad50 form discrete nuclear foci in response to treatment with DSB-inducing agents but not in response to UV irradiation. hMre11 and hRad50 foci colocalize after treatment with ionizing radiation and are distinct from those of the DSB repair protein, hRad51. Our data indicate that an irradiated cell is competent to form either hMre11-hRad50 foci or hRad51 foci, but not both. The multiplicity of hMre11 and hRad50 foci is much higher in the DSB repair-deficient cell line 180BR than in repair-proficient cells. hMre11-hRad50 focus formation is markedly reduced in cells derived from ataxia-telangiectasia patients, whereas hRad51 focus formation is markedly increased. These experiments support genetic evidence from Saccharomyces cerevisiae indicating that Mre11-Rad50 have roles distinct from that of Rad51 in DSB repair. Further, these data indicate that hMre11-hRad50 foci form in response to DNA DSBs and are dependent upon a DNA damage-induced signaling pathway.

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