FANCM regulates repair pathway choice at stalled replication forks

Panday, Arvind; Willis, Nicholas A.; Elango, Rajula; Menghi, Francesca; Duffey, Erin E.; Liu, Edison T.; Scully, Ralph

doi:10.1016/j.molcel.2021.03.044

Cited by 47 publications

(33 citation statements)

References 94 publications

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“…In the SCR-RFP reporter, site-specific stalling of replication forks induced GFP − RFP + cells, which represent excess ~10 kb MH-mediated TDs 24 . However, both spontaneous and I-SceI-induced GFP − RFP + cells are products from GFP repeat-mediated TD, containing three copies of GFP with a ~10-kb duplication span 48 , 49 . To determine which type of TDs represented by GFP − RFP + cells is induced by DNA nicks and could be promoted by BRCA1 deficiency, we first analyzed the frequencies of GFP − RFP + cells induced spontaneously or by nCas9 and Cas9 (Fig.…”

Section: Resultsmentioning

confidence: 99%

DNA nicks induce mutational signatures associated with BRCA1 deficiency

et al. 2022

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Analysis of human cancer genome sequences has revealed specific mutational signatures associated with BRCA1-deficient tumors, but the underlying mechanisms remain poorly understood. Here, we show that one-ended DNA double strand breaks (DSBs) converted from CRISPR/Cas9-induced nicks by DNA replication, not two-ended DSBs, cause more characteristic chromosomal aberrations and micronuclei in Brca1-deficient cells than in wild-type cells. BRCA1 is required for efficient homologous recombination of these nick-converted DSBs and suppresses bias towards long tract gene conversion and tandem duplication (TD) mediated by two-round strand invasion in a replication strand asymmetry. However, aberrant repair of these nick-converted one-ended DSBs, not that of two-ended DSBs in Brca1-deficient cells, generates mutational signatures such as small indels with microhomology (MH) at the junctions, translocations and small MH-mediated TDs, resembling those in BRCA1-deficient tumors. These results suggest a major contribution of DNA nicks to mutational signatures associated with BRCA1 deficiency in cancer and the underlying mechanisms.

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Section: Resultsmentioning

confidence: 99%

DNA nicks induce mutational signatures associated with BRCA1 deficiency

et al. 2022

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“…It has been recently shown that loss-of-function mutations in Fancm and Brca1 leads to synthetic lethality ( Panday et al, 2021 ). Like SMARCAL1, FANCM and BRCA1 are also required for repair of stalled replication fork.…”

Section: Discussionmentioning

confidence: 99%

On the Interaction Between SMARCAL1 and BRG1

Bisht

Patne

Radhakrishnan

et al. 2022

Front. Cell Dev. Biol.

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SMARCAL1 and BRG1, both classified as ATP-dependent chromatin remodeling proteins, play a role in double-strand break DNA damage response pathways. Mutations in SMARCAL1 cause Schimke Immuno-osseous Dysplasia (SIOD) while mutations in BRG1 are associated with Coffin-Siris Syndrome (CSS4). In HeLa cells, SMARCAL1 and BRG1 co-regulate the expression of ATM, ATR, and RNAi genes on doxorubicin-induced DNA damage. Both the proteins are found to be simultaneously present on the promoter of these genes. Based on these results we hypothesized that SMARCAL1 and BRG1 interact with each other forming a complex. In this paper, we validate our hypothesis and show that SMARCAL1 and BRG1 do indeed interact with each other both in the absence and presence of doxorubicin. The formation of these complexes is dependent on the ATPase activity of both SMARCAL1 and BRG1. Using deletion constructs, we show that the HARP domains of SMARCAL1 mediate interaction with BRG1 while multiple domains of BRG1 are probably important for binding to SMARCAL1. We also show that SIOD-associated mutants fail to form a complex with BRG1. Similarly, CSS4-associated mutants of BRG1 fail to interact with SMARCAL1, thus, possibly contributing to the failure of the DNA damage response pathway and pathophysiology associated with SIOD and CSS4.

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“… Note: In this protocol to study the recruitment of protein in response to site-specific stalled fork and site-specific DNA double-strand break (DSB), we used a reporter system. This system uses Escherichia coli Tus/ Ter replication fork barrier (RFB) ( Berghuis et al., 2015 ; Elshenawy et al., 2015 ) and also contains the I-SceI rare-cutting homing endonuclease cut site that generates site-specific double-strand break and targeted as a single copy to the Rosa26 locus of mouse chromosome 6 in mES cells ( Panday et al., 2021 ; Willis et al., 2017 , 2018 ). Therefore, this reporter is an ideal system to parallelly study and compare the site-specific recruitment of proteins by ChIP-qPCR and Cut&RUN-qPCR in response to stalled replication fork and DSB ( Figure 1 ) Thaw mES cells into a single well of a six-well plate pre-coated with mouse embryonic fibroblast (MEF) “feeders” cells that have been mitotically inactivated by lethal irradiation.…”

Section: Before You Beginmentioning

confidence: 99%