scholarly journals Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand crosslinks

2019 ◽  
Author(s):  
Kimberly A. Rickman ◽  
Ray Noonan ◽  
Francis P. Lach ◽  
Sunandini Sridhar ◽  
Anderson T. Wang ◽  
...  
Keyword(s):  
Fanconi Anemia ◽  
Essential Gene ◽  
Dna Lesions ◽  
Replication Forks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Interstrand Crosslinks ◽  
Pathogenic Variants ◽  
Dna Interstrand Crosslinks ◽  
Stalled Replication Forks

SummaryDNA interstrand crosslinks (ICLs) are a form of DNA damage that requires the interplay of a number of repair proteins including those of the Fanconi anemia (FA) and the homologous recombination (HR) pathways. Pathogenic variants in the essential gene BRCA2/FANCD1, when monoallelic, predispose to breast and ovarian cancer, and when biallelic, results in a severe subtype of Fanconi anemia. BRCA2 function in the FA pathway is attributed to its role as a mediator of the RAD51 recombinase in HR repair of the programmed DNA double strand breaks (DSB). BRCA2 and RAD51 functions are also required to protect stalled replication forks from nucleolytic degradation during response to hydroxyurea (HU). While RAD51 has been shown to be necessary in the early steps of ICL repair to prevent aberrant nuclease resection, the role of BRCA2 in this process has not been described. Here, based on the analysis of BRCA2 DNA binding domain (DBD) mutants discovered in FA patients presenting with atypical FA-like phenotypes, we establish that BRCA2 is necessary for protection of DNA at an ICL. Cells carrying DBD BRCA2 mutations are sensitive to ICL inducing agents but resistant to HU treatment consistent with relatively high HR repair in these cells. BRCA2 function at an ICL protects against DNA2-WRN nuclease-helicase complex and not the MRE11 nuclease implicated in the resection of HU-stalled replication forks. Our results also indicate that unlike the processing at HU-stalled forks, function of the SNF2 translocases (SMARCAL1, ZRANB3, or HLTF), implicated in fork reversal, are not an integral component of the ICL repair, pointing to a different mechanism of fork protection at different DNA lesions.

scholarly journals The homologous recombination complex of the Rad51 paralogs Rad55-Rad57 avoids translesion DNA polymerase recruitment and counterbalances mutagenesis induced by UV radiation

2021 ◽  
Author(s):  
Laurent G Maloisel ◽  
Emilie Ma ◽  
Eric Coic
Keyword(s):  
Homologous Recombination ◽  
Uv Radiation ◽  
Dna Lesions ◽  
Template Switching ◽  
Dna Double Strand Breaks ◽  
Dna Damage Tolerance ◽  
Strand Breaks ◽  
Rad51 Paralogs ◽  
Replicative Polymerases ◽  
Stalled Replication Forks

Bypass of DNA lesions that block replicative polymerases during DNA replication relies on several DNA damage tolerance pathways. The error-prone translesion synthesis (TLS) pathway involves specialized DNA polymerases that incorporate nucleotides in front of base lesions. The template switching and the homologous recombination (HR) pathways are mostly error-free because the bypass is performed by using typically the sister chromatid as a template. This is promoted by the Rad51 recombinase that forms nucleoprotein filaments on single-strand DNA (ssDNA). The balance between error-prone and error-free pathways controls the level of mutagenesis. In yeast, the Rad55-Rad57 complex of Rad51 paralogs is required for Rad51 filament formation and stability, notably by counteracting the Srs2 antirecombinase. Several reports showed that Rad55-Rad57 promotes HR at stalled replication forks more than at DNA double-strand breaks (DSB), suggesting that this complex is more efficient at ssDNA gaps and thus, could control the recruitment of TLS polymerases. To address this point, we studied the interplay between Rad55-Rad57 and the TLS polymerases Polζ and Polη following UV radiation. We confirmed that Rad55-Rad57 protects Rad51 filaments from Srs2 dismantling activity but we found that it is also essential for the promotion of UV-induced HR independently of Srs2. In addition, we observed that cell UV sensitivity, but not DSB sensitivity, is synergistically increased when Rad55 and Polζ deletions are combined. Moreover, we found that mutagenesis and HR frequency were increased in rad55∆ mutants and in TLS-deficient cells, respectively. Finally, UV-induced HR was partially restored in Rad55-deficient cells with mutated Polζ or Polη. Overall, our data suggest that the HR and TLS pathways compete for the same ssDNA substrates and that the Rad55-Rad57 complex of Rad51 paralogs prevents the recruitment of TLS polymerases and counterbalances mutagenesis.

scholarly journals Ablation of PARP-1 does not interfere with the repair of DNA double-strand breaks, but compromises the reactivation of stalled replication forks

Oncogene ◽  
2004 ◽  
Vol 23 (21) ◽  
pp. 3872-3882 ◽  
Author(s):  
Yun-Gui Yang ◽  
Ulrich Cortes ◽  
Srinivas Patnaik ◽  
Maria Jasin ◽  
Zhao-Qi Wang
Keyword(s):  
Double Strand Breaks ◽  
Replication Forks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Stalled Replication Forks ◽  
Parp 1

scholarly journals DNA clamp function of the mono-ubiquitinated Fanconi Anemia FANCI-FANCD2 complex

2019 ◽  
Author(s):  
Renjing Wang ◽  
Shengliu Wang ◽  
Ankita Dhar ◽  
Christopher Peralta ◽  
Nikola P. Pavletich
Keyword(s):  
Fanconi Anemia ◽  
Translesion Synthesis ◽  
The Other ◽  
Replication Forks ◽  
Dna Structures ◽  
Cancer Predisposition Syndrome ◽  
Interstrand Crosslinks ◽  
Branched Dna ◽  
Closed Ring ◽  
Dna Interstrand Crosslinks

ABSTRACTThe FANCI-FANCD2 (ID) complex, mutated in the Fanconi Anemia (FA) cancer predisposition syndrome, is required for the repair of replication forks stalled at DNA interstrand crosslinks (ICL) and related lesions1. The FA pathway is activated when two replication forks converge onto an ICL2, triggering the mono-ubiquitination of the ID complex. ID mono-ubiquitination is essential for ICL repair by excision, translesion synthesis and homologous recombination, but its function was hitherto unknown1,3. Here, the 3.48 Å cryo-EM structure of mono-ubiquitinated ID (IDUb) bound to DNA reveals that it forms a closed ring that encircles the DNA. Compared to the cryo-EM structure of the non-ubiquitinated ID complex bound to ICL DNA, described here as well, mono-ubiquitination triggers a complete re-arrangement of the open, trough-like ID structure through the ubiquitin of one protomer binding to the other protomer in a reciprocal fashion. The structures, in conjunction with biochemical data, indicate the mono-ubiquitinated ID complex looses its preference for ICL and related branched DNA structures, becoming a sliding DNA clamp that can coordinate the subsequent repair reactions. Our findings also reveal how mono-ubiquitination in general can induce an alternate structure with a new function.

scholarly journals Chromosome Instability in Fanconi Anemia: From Breaks to Phenotypic Consequences

Genes ◽  
2020 ◽  
Vol 11 (12) ◽  
pp. 1528
Author(s):  
Benilde García-de-Teresa ◽  
Alfredo Rodríguez ◽  
Sara Frias
Keyword(s):  
Dna Repair ◽  
Fanconi Anemia ◽  
Chromosomal Instability ◽  
Bone Marrow Failure ◽  
Chromosome Instability ◽  
Premature Aging ◽  
Dna Double Strand Breaks ◽  
Interstrand Crosslinks ◽  
Pathogenic Variants ◽  
Extreme Risk

Fanconi anemia (FA), a chromosomal instability syndrome, is caused by inherited pathogenic variants in any of 22 FANC genes, which cooperate in the FA/BRCA pathway. This pathway regulates the repair of DNA interstrand crosslinks (ICLs) through homologous recombination. In FA proper repair of ICLs is impaired and accumulation of toxic DNA double strand breaks occurs. To repair this type of DNA damage, FA cells activate alternative error-prone DNA repair pathways, which may lead to the formation of gross structural chromosome aberrations of which radial figures are the hallmark of FA, and their segregation during cell division are the origin of subsequent aberrations such as translocations, dicentrics and acentric fragments. The deficiency in DNA repair has pleiotropic consequences in the phenotype of patients with FA, including developmental alterations, bone marrow failure and an extreme risk to develop cancer. The mechanisms leading to the physical abnormalities during embryonic development have not been clearly elucidated, however FA has features of premature aging with chronic inflammation mediated by pro-inflammatory cytokines, which results in tissue attrition, selection of malignant clones and cancer onset. Moreover, chromosomal instability and cell death are not exclusive of the somatic compartment, they also affect germinal cells, as evidenced by the infertility observed in patients with FA.

DNA interstrand crosslinks arising from DAN strand breaks at true abasic sites in duplex DNA

2018 ◽  
Author(s):  
◽  
Zhiyu Yang
Keyword(s):  
Dna Lesions ◽  
University Of Missouri ◽  
Strand Breaks ◽  
Interstrand Crosslinks ◽  
Abasic Sites ◽  
Single Strand Breaks ◽  
Dna Interstrand Crosslinks ◽  
Ap Sites ◽  
Dna Strand

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Abasic sites (apurinic/apyrimidinic sites, Ap sites) are one of the most common forms of lesions found in genomic DNA. For the past decade, Gates group has endeavored to identify DNA interstrand crosslinks arising from Ap sites, that are sources of endogenous interstrand crosslinks which relate to aging, cancer and neurodegenerative diseases. This thesis describes my work in this area, starting from studying the DNA strand cleavage at abasic sites via [beta]-elimination reaction, that can generate a family of structurally diverse sugar remnants at the 3’ terminus of the nicked strand under physiological conditions. Using an in vitro system, I have characterized a series of DNA interstrand crosslinks arising from these single strand breaks, which structures are dependent on changing DNA sequence contexts and varied assay conditions that are all bio-relevant. These structures are novel and haven’t been reported to the best of our knowledge. In the meantime, this work has highlighted amine catalysis in Michael addition, and brought to attention the role of GSH in the formation of complex DNA lesions.

scholarly journals Ionizing Radiation–dependent γ-H2AX Focus Formation Requires Ataxia Telangiectasia Mutated and Ataxia Telangiectasia Mutated and Rad3-related

2005 ◽  
Vol 16 (5) ◽  
pp. 2566-2576 ◽  
Author(s):  
Joanna D. Friesner ◽  
Bo Liu ◽  
Kevin Culligan ◽  
Anne B. Britt
Keyword(s):  
Ionizing Radiation ◽  
Ataxia Telangiectasia ◽  
Double Strand Breaks ◽  
Replication Forks ◽  
Dna Double Strand Breaks ◽  
Ataxia Telangiectasia Mutated ◽  
Focus Formation ◽  
Strand Breaks ◽  
M Phase ◽  
Stalled Replication Forks

The histone variant H2AX is rapidly phosphorylated at the sites of DNA double-strand breaks (DSBs). This phosphorylated H2AX (γ-H2AX) is involved in the retention of repair and signaling factor complexes at sites of DNA damage. The dependency of this phosphorylation on the various PI3K-related protein kinases (in mammals, ataxia telangiectasia mutated and Rad3-related [ATR], ataxia telangiectasia mutated [ATM], and DNA-PKCs) has been a subject of debate; it has been suggested that ATM is required for the induction of foci at DSBs, whereas ATR is involved in the recognition of stalled replication forks. In this study, using Arabidopsis as a model system, we investigated the ATR and ATM dependency of the formation of γ-H2AX foci in M-phase cells exposed to ionizing radiation (IR). We find that although the majority of these foci are ATM-dependent, ∼10% of IR-induced γ-H2AX foci require, instead, functional ATR. This indicates that even in the absence of DNA replication, a distinct subset of IR-induced damage is recognized by ATR. In addition, we find that in plants, γ-H2AX foci are induced at only one-third the rate observed in yeasts and mammals. This result may partly account for the relatively high radioresistance of plants versus yeast and mammals.

scholarly journals Defective Ribonucleoside Diphosphate Reductase Impairs Replication Fork Progression in Escherichia coli

2007 ◽  
Vol 189 (9) ◽  
pp. 3496-3501 ◽  
Author(s):  
Estrella Guarino ◽  
Alfonso Jiménez-Sánchez ◽  
Elena C. Guzmán
Keyword(s):  
Replication Fork ◽  
Double Strand Breaks ◽  
Permissive Temperature ◽  
Replication Forks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Replication Fork Progression ◽  
Holliday Junction Resolvase ◽  
Ribonucleoside Diphosphate Reductase ◽  
Stalled Replication Forks

ABSTRACT The observed lengthening of the C period in the presence of a defective ribonucleoside diphosphate reductase has been assumed to be due solely to the low deoxyribonucleotide supply in the nrdA101 mutant strain. We show here that the nrdA101 mutation induces DNA double-strand breaks at the permissive temperature in a recB-deficient background, suggesting an increase in the number of stalled replication forks that could account for the slowing of replication fork progression observed in the nrdA101 strain in a Rec+ context. These DNA double-strand breaks require the presence of the Holliday junction resolvase RuvABC, indicating that they have been generated from stalled replication forks that were processed by the specific reaction named “replication fork reversal.” Viability results supported the occurrence of this process, as specific lethality was observed in the nrdA101 recB double mutant and was suppressed by the additional inactivation of ruvABC. None of these effects seem to be due to the limitation of the deoxyribonucleotide supply in the nrdA101 strain even at the permissive temperature, as we found the same level of DNA double-strand breaks in the nrdA + strain growing under limited (2-μg/ml) or under optimal (5-μg/ml) thymidine concentrations. We propose that the presence of an altered NDP reductase, as a component of the replication machinery, impairs the progression of the replication fork, contributing to the lengthening of the C period in the nrdA101 mutant at the permissive temperature.

scholarly journals Protection of nascent DNA at stalled replication forks is mediated by phosphorylation of RIF1 intrinsically disordered region

2021 ◽  
Author(s):  
Sandhya Balasubramanian ◽  
Matteo Andreani ◽  
Júlia Goncalves Andrade ◽  
Tannishtha Saha ◽  
Javier Garzón ◽  
...  
Keyword(s):  
Replication Stress ◽  
Replication Forks ◽  
Dna Double Strand Breaks ◽  
Protective Function ◽  
Multifunctional Protein ◽  
Strand Breaks ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Region ◽  
Dna Replication Stress ◽  
Stalled Replication Forks

RIF1 is a multifunctional protein that plays key roles in the regulation of DNA processing. During repair of DNA double-strand breaks (DSBs), RIF1 functions in the 53BP1-Shieldin pathway that inhibits resection of DNA ends to modulate the cellular decision on which repair pathway to engage. Under conditions of replication stress, RIF1 protects nascent DNA at stalled replication forks from degradation by the DNA2 nuclease. How these RIF1 activities are regulated at the post-translational level has not yet been elucidated. Here, we identified a cluster of conserved ATM/ATR consensus SQ motifs within the intrinsically disordered region (IDR) of mouse RIF1 that are phosphorylated in proliferating B lymphocytes. We found that phosphorylation of the conserved IDR SQ cluster is dispensable for the inhibition of DSB resection by RIF1, but is essential to counteract DNA2-dependent degradation of nascent DNA at stalled replication forks. Therefore, our study identifies a key molecular switch that enables the genome-protective function of RIF1 during DNA replication stress.

scholarly journals Chromosome Instability in Fanconi Anemia: From Breaks to Phenotypic Consequences

2020 ◽  
Author(s):  
Benilde García-de-Teresa ◽  
Alfredo Rodríguez ◽  
Sara Frias
Keyword(s):  
Dna Repair ◽  
Fanconi Anemia ◽  
Chromosomal Instability ◽  
Bone Marrow Failure ◽  
Chromosome Instability ◽  
Premature Aging ◽  
Dna Double Strand Breaks ◽  
Interstrand Crosslinks ◽  
Pathogenic Variants ◽  
Extreme Risk

Abstract: Fanconi anemia (FA), a chromosomal instability syndrome, is caused by inherited pathogenic variants in any of 22 FANC genes, that cooperate in the FA/BRCA pathway. This pathway regulates the repair of DNA interstrand crosslinks (ICLs) through homologous recombination. In FA proper repair of ICLs is impaired, and accumulation of toxic DNA double strand breaks occurs. In order to repair this type of DNA damage, FA cells activate alternative error-prone DNA repair pathways, that may lead to the formation of gross structural chromosome aberrations of which radial figures are the hallmark of FA and their segregation during cell division are the origin of subsequent aberrations like translocations, dicentrics and acentric fragments. The deficiency in DNA repair has pleiotropic consequences in the phenotype of patients with FA, including developmental alterations, bone marrow failure and an extreme risk to develop cancer. The mechanisms leading to the physical abnormalities during embryonic development have not been clearly elucidated, however FA has features of premature aging with chronic inflammation mediated by pro-inflammatory cytokines, that results in tissue attrition, selection of malignant clones and cancer onset. Moreover, the effect of the FA/BRCA pathway in germinal cells, evidenced by infertility in patients with FA attests of chromosomal instability and cell death also occurring in the germinal compartment.

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