Role of RTEL1 helicase in the regulation of homologous recombination during DNA replication

Abstract

Homologous recombination (HR) plays an essential role in the repair of DNA double-strand breaks (DSBs), replication stress responses, and genome maintenance. Although the mechanism of HR has been extensively studied in the context of DSBs, recent evidence suggests that HR plays a crucial role during DNA replication, both physiologically and under stress. Dysregulation of HR leads to chromosomal instability characterized by the under-replicated genome, increased somatic mutations, chromosomal translocations, deletions, amplifications, and loss of heterozygosity (LOH). Unregulated HR during replication leads to gross chromosomal rearrangements that can impair genome duplication and compromise genome stability. The intricate link between HR and genome stability is highlighted by the fact that various HR proteins, including BRCA1, BRCA2, RAD51, and RAD51 paralogs, are implicated in cancer development. Cells have evolved with multiple layers of regulation to maintain the fidelity of HR. A specialized group of proteins called anti-recombinases offers quality control mechanisms to facilitate RAD51 filament disassembly and prevent downstream HR events. The multi-pronged regulation of HR involves maintaining the balance of pro- and anti-recombinases in check to avoid excess, untimely, and aberrant recombination at stalled replication forks. The RTEL1, PARI, FBH1, BLM, RECQL5, and FIGNL1 proteins have been implicated as potential anti-recombinases. RTEL1 helicase has been shown to suppress hyper-recombination during DSB repair. It is unclear whether RTEL1 helicase regulates HR during DNA replication. We uncovered a novel role of RTEL1 in regulating RAD51-mediated HR and fork reversal. The absence of RTEL1 led to cell survival defects upon HU-induced replication stress but not with DSB-inducing zeocin, highlighting a specific role of RTEL1 in dealing with replication problems in the genome. In addition, the loss of RTEL1 was found to be associated with genome-wide replication defects such as slow-moving replication forks, fork asymmetry, fork stalling, and fork restart defects. iPOND analysis revealed that RTEL1 helicase, RAD51, and RAD51 paralogs are enriched stalled replication sites, underscoring a dynamic assembly of RTEL1 and RAD51 paralogs at stalled replication forks. The loss of RTEL1 resulted in hyper-recombination at I-SceI-induced DSBs and stalled fork sites, suggesting that RTEL1 suppresses HR during DNA replication. Importantly, the hyper-recombination phenotype and replication defects in RTEL1-depleted cells were found to be mitigated by co-depletion of RAD51 and RAD51 paralogs. This highlights the important role of RTEL1 in regulating HR at replicating sites to facilitate global DNA replication, promoting genome stability. Notably, co-depletion of fork remodelers such as SMARCAL1/ZRANB3/HLTF/FBH1 was found to rescue replication defects in RTEL1-deficient cells. In addition, the expression of HR and fork reversal defective RAD51 mutants were also found to rescue replication defects in RTEL1-depleted cells. This suggests that stalled forks in RTEL1-depleted cells undergo RAD51-mediated fork reversal, contributing to reduced replication rates. Using iPOND, ChIP, and IF experiments, it was shown that in the absence of RTEL1, there is a persistent accumulation of RAD51 and RAD51 paralogs at the stalled fork sites. Mechanistically, it suggests that RTEL1 prevented the accumulation of RAD51 and RAD51 paralogs at stalled fork sites, thus suppressing excessive RAD51-mediated HR and fork reversal to facilitate genome duplication. Additionally, it was shown that RTEL1 PCNA interaction and its helicase activity are required for suppressing hyperrecombination to facilitate error-free genome duplication. Collectively, our data suggest that RTEL1 suppresses RAD51-mediated aberrant HR activity and prevents excessive fork remodeling during DNA replication to promote error-free genome duplication.