CtIP-Mediated Fork Protection Synergizes with BRCA1 to Suppress Genomic Instability upon DNA Replication Stress

Przetocka, Sara; Porro, Antônio; Bolck, Hella Anna; Walker, Christina; Lezaja, Aleksandra; Trenner, Anika; Aesch, Christine von; Himmels, Sarah-Felicitas; D’Andrea, Alan D.; Ceccaldi, Raphaël; Altmeyer, Matthias; Sartori, Alessandro A.

doi:10.1016/j.molcel.2018.09.014

Cited by 99 publications

(100 citation statements)

References 62 publications

(94 reference statements)

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“…While the role of MRE11 in nascent strand degradation of BRCA2 deficient cells has been widely shown, there is conflicting data about resection mediated by DNA2 [25,26,50]. A role for DNA2 with WRN in replication fork restart has been described, and it has also been reported that DNA2 degrades nascent DNA at stalled forks in the setting of RECQ1, BOD1L, or Abro1 deficiency [24,[51][52][53].…”

Section: Brca2 Function At the Hu-stalled Replication Forkmentioning

confidence: 99%

“…Recent reports have also implicated EXO1 and CTIP as degrading HU-stalled forks in the absence of BRCA2 [25]. Conversely, CTIP has been reported to be required to restrain DNA2 activity at stalled replication forks in the absence of BRCA1/2 [26].…”

Section: Brca2 Function At the Hu-stalled Replication Forkmentioning

confidence: 99%

“…Outside of their role in HR and ICL repair, BRCA2 and RAD51 along with a number of other recently described proteins, function in replication fork protection [24]. In the absence of replication fork protection, newly synthesized DNA is degraded at hydroxyurea (HU) stalled replication forks and a number of nucleases including MRE11, CTIP, and EXO1 have been implicated in the process [24][25][26].…”

Section: Introductionmentioning

confidence: 99%

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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: Brca2 Function At the Hu-stalled Replication Forkmentioning

confidence: 99%

Section: Brca2 Function At the Hu-stalled Replication Forkmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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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“…Protecting stalled forks from excessive nucleolytic resection is critical in resolving replication stress and maintaining genome stability. A number of studies have shown that many homology-directed repair (HDR) factors, including BRCA1/2, RAD51, CtIP, have HDRindependent roles in protecting fork stability (Hashimoto et al, 2010;Kolinjivadi et al, 2017;Przetocka et al, 2018;Taglialatela et al, 2017). In this study, we provide direct evidence that the ssDNA-binding complex CST is a new component at stalled forks and functions in limiting excessive nascent strand degradation.…”

Section: Discussionmentioning

confidence: 78%

Human CST complex protects replication fork stability by directly blocking MRE11 degradation of nascent strand DNA

Shiva

et al. 2019

Preprint

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Degradation and collapse of stalled replication forks are main sources of genome instability, yet the molecular mechanism for protecting forks from degradation/collapse is not well understood. Here, we report that human CST (CTC1-STN1-TEN1), a single-stranded DNA binding protein complex, localizes at stalled forks and protects forks from MRE11 nuclease degradation upon replication perturbation. CST deficiency causes nascent strand degradation, ssDNA accumulation after fork stalling, and delay in replication recovery, leading to cellular sensitivity to fork stalling agents. Purified CST binds to 5' overhangs and directly blocks MRE11 degradation in vitro, and the DNA binding ability of CST is required for blocking MRE11-mediated nascent strand degradation. Finally, we uncover that CST and BRCA2 form non-overlapping foci upon fork stalling, and CST inactivation is synthetic with BRCA2 deficiency in inducing genome instability. Collectively, our findings identify CST as an important fork protector to preserve genome integrity under replication perturbation.

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“…The crystal structure of the RAD51:BRC8-2 complex has provided novel insight into the BRC repeat binding to RAD51, in particular in identification of the extended β-hairpin formed by the FxxA module and the critical role Ser2056 BRC8 in facilitating the formation of this structure. The enhanced affinity of BRC8-2 may render it an attractive tool in studies that seek to investigate the effect of disrupting the RAD51:BRCA2 interaction using cell penetrating peptide derivatives of BRC repeats 36,39 . We were able to demonstrate the utility of BRC8-2 in a functional cellular assay for disruption of radiation-induced RAD51 foci formation.…”

Section: Discussionmentioning

confidence: 99%

Improved RAD51 binders through motif shuffling based on the modularity of BRC repeats

Lindenburg

Pantelejevs

Gielen

et al. 2020

Preprint

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Exchanges of protein sequence modules support leaps in function unavailable through point mutations during evolution. Here we study the role of the two RAD51-interacting modules within the eight binding BRC repeats of BRCA2. We created 64 chimeric repeats by shuffling these modules and measured their binding to RAD51. We found that certain shuffled repeats were stronger than any of the natural repeats, suggesting balancing of relative properties in BRC repeats. Surprisingly, the contribution from the two modules was poorly correlated with affinities of natural repeats, with weak BRC8 repeat containing the most effective N-terminal module. The binding of the strongest chimera, BRC8-2, to RAD51 was improved by -2.44 kCal/mol compared to the strongest natural repeat, BRC4. Crystal structure of RAD51:BRC8-2 complex shows an improved interface fit and an extended -hairpin in this repeat. BRC8-2 was shown to function in human cells, preventing the formation of nuclear foci after ionizing radiation.

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CtIP-Mediated Fork Protection Synergizes with BRCA1 to Suppress Genomic Instability upon DNA Replication Stress

Abstract: Highlights d CtIP protects reversed forks from nucleolytic degradation d CtIP prevents DNA2-dependent over-resection of nascent strands d CtIP nuclease mutants are defective in fork protection d CtIP-mediated fork protection synergizes with BRCA1, not BRCA2

Cited by 99 publications

References 62 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

Human CST complex protects replication fork stability by directly blocking MRE11 degradation of nascent strand DNA

Improved RAD51 binders through motif shuffling based on the modularity of BRC repeats

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