RADX Modulates RAD51 Activity to Control Replication Fork Protection

Bhat, Kamakoti P.; Krishnamoorthy, Archana; Dungrawala, Huzefa; Garcin, Edwige B.; Modesti, Mauro; Chen, David

doi:10.1016/j.celrep.2018.06.061

Cited by 99 publications

(110 citation statements)

References 28 publications

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“…The previously described RAD51/FANCR p.T131P patient-derived cell line that is proficient for HR but defective in ICL repair displays hyperactivation of RPA upon MMC treatment [27]. Given that the interaction of BRCA2 and RAD51 is required for their canonical function in HR and their non-canonical function in replication fork protection at HU-stalled forks, we investigated whether BRCA2 also functions in preventing increased ssDNA generation at ICLs [27][28][29][30]. We observed an increase in RPA foci formation and RPA phosphorylation in HSC62 MUT cells compared to wild type fibroblasts upon MMC treatment (Figure 2G, Figure S3A).…”

Section: Increased Rpa Activation In Brca2 Dbd Variants Is Dependent mentioning

confidence: 99%

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

Rickman

Noonan

Lach

et al. 2019

Preprint

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DNA 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.2 Introduction:

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Section: Increased Rpa Activation In Brca2 Dbd Variants Is Dependent mentioning

confidence: 99%

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

Rickman

Noonan

Lach

et al. 2019

Preprint

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“…Recently, the OB-fold containing protein RADX has been identified to control replication fork protection by antagonizing RAD51 binding to single stranded DNA (Bhat et al, 2018;Dungrawala et al, 2017;Schubert et al, 2017). In this paper, we explore the telomere protein composition by proximity-dependent labeling using TRF1, TRF2 and POT1 as baits.…”

Section: Introductionmentioning

confidence: 99%

RADX Sustains POT1 Function at Telomeres to Counteract RAD51 Binding, which Triggers Telomere Fragility

Briod

Glousker

Lingner

2020

Preprint

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The 3' terminal DNA extensions at chromosome ends can become engaged into multiple biochemical reactions during DNA replication, telomerase-mediated telomere extension, homology directed DNA repair, nucleolytic processing and DNA damage checkpoint activation. To keep these activities in check, telomeric 3' overhangs can be hidden in t-loop structures or they associate with specialized proteins such as POT1. Here, we explore the telomeric microenvironment using a proximity-dependent labeling approach and identify the oligonucleotide/oligosaccharide-binding (OB)-fold containing protein RADX. RADX binds single-stranded telomeric DNA throughout the cell cycle along with POT1, suppressing accumulation of fragile telomeres, which are indicative of telomere replication defects.Telomere fragility in POT1 and RADX double-depleted cells was due to accumulation of the RAD51 recombinase at telomeres. RADX also supports DNA damage signaling at POT1depleted telomeres counteracting RAD51 binding. Thus, RADX represents next to POT1 a second OB-fold containing single-strand telomere binding protein sustaining telomere protection.

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“…HR reactions mediated by Rad51 (9)(10)(11)(12) and modulated by the Rad51 paralogues (13) are also required for resolving DNA replication impediments, by promoting protection and restart of stalled replication forks during replication stress. An even more intimate association between HR and DNA replication has been described in bacteria and archaea, where RecA (14)(15)(16)(17) and RadA (18) can mediate DNA replication when origins (the genome sites where DNA synthesis begins during replication) have been removed.…”

Section: Introductionmentioning

confidence: 99%

Conditional knockout of RAD51-related genes inLeishmania majorreveals a critical role for homologous recombination during genome replication

Damasceno

Reis-Cunha

Crouch

et al. 2019

Preprint

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Homologous recombination (HR) has an intimate relationship with genome replication, both during repair of DNA lesions that might prevent DNA synthesis and in tackling stalls to the replication fork. Recent studies led us to ask if HR might have a more central role in replicating the genome of Leishmania, a eukaryotic parasite. Conflicting evidence has emerged regarding whether or not HR genes are essential, and genomewide mapping has provided evidence for an unorthodox organisation of DNA replication initiation sites, termed origins. To answer this question, we have employed a combined CRISPR/Cas9 and DiCre approach to rapidly generate and assess the effect of conditional ablation of RAD51 and three RAD51-related proteins in Leishmania major. Using this approach, we demonstrate that loss of any of these HR factors is not immediately lethal, but in each case growth slows with time and leads to DNA damage, accumulation of cells with aberrant DNA content, and genome-wide mutation. Despite these similarities, we show that only loss of RAD51 and RAD51-3 impairs DNA synthesis, and that the factors act in distinct ways. Finally, we reveal that loss of RAD51 has a profound effect on DNA replication, causing loss of initiation at the major origins and increased DNA synthesis at subtelomeres. Our work clarifies questions regarding the importance of HR to survival of Leishmania and reveals an unanticipated, central role for RAD51 in the programme of genome replication in a microbial eukaryote.

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RADX Modulates RAD51 Activity to Control Replication Fork Protection

Cited by 99 publications

References 28 publications

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

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

RADX Sustains POT1 Function at Telomeres to Counteract RAD51 Binding, which Triggers Telomere Fragility

Conditional knockout of RAD51-related genes inLeishmania majorreveals a critical role for homologous recombination during genome replication

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