Abstract
Homologous recombination (HR) factors were recently implicated in DNA replication fork remodeling and protection. While maintaining genome stability, HR-mediated fork remodeling promotes cancer chemoresistance, by as-yet elusive mechanisms. Five HR cofactors – the RAD51 paralogs RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3 – recently emerged as crucial tumor suppressors. Albeit extensively characterized in DNA repair, their role in replication has not been addressed systematically. Here, we identify all RAD51 paralogs while screening for modulators of RAD51 recombinase upon replication stress. Single-molecule analysis of fork progression and architecture in isogenic cellular systems shows that the BCDX2 subcomplex restrains fork progression upon stress, promoting fork reversal. Accordingly, BCDX2 primes unscheduled degradation of reversed forks in BRCA2-defective cells, boosting genomic instability. Conversely, the CX3 subcomplex is dispensable for fork reversal, but mediates efficient restart of reversed forks. We propose that RAD51 paralogs sequentially orchestrate clinically relevant transactions at replication forks, cooperatively promoting fork remodeling and restart.
Highlights
Homologous recombination (HR) factors were recently implicated in DNA replication fork remodeling and protection
The recent discoveries that mutations in classical RAD51 paralog genes predispose to cancer and Fanconi anemia[47,48,49,50,51,52,53] encouraged new mechanistic studies on these HR accessory factors, focused on the human proteins and their possible role in the replication stress response
Until very recently, genetic investigations on these proteins were largely limited to single small interfering RNA (siRNA) or mutated cell lines in specific model systems, calling for more systematic genetic investigations of these proteins in isogenic human systems, with respect to their role upon replication stress
Summary
Homologous recombination (HR) factors were recently implicated in DNA replication fork remodeling and protection. Other HR factors—like BRCA1 and BRCA2—protect stalled forks from excessive nucleolytic degradation by promoting efficient RAD51 loading[9,17,18] This function in replication stress is genetically uncoupled from their canonical role in DSB repair[14,17,19], is associated with the exquisite chemosensitivity of BRCA-deficient cells[20] and has been recently extended to additional HR factors[21]. This deregulated degradation of nascent DNA is genetically dependent on fork remodeling, as regressed arms are mandatory entry points for DNA degrading enzymes[13,14,22,23]
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