Understanding the role of the RECQL5 helicase in regulating homologous recombination during DNA replication and genome maintenance

Abstract

Faithful genome duplication is essential for maintaining genomic integrity and cell viability. During each cell cycle, the DNA replication machinery must accurately duplicate the genome. However, replication forks frequently encounter barriers that impede their progression, leading to fork stalling and replication stress. Fork reversal is a key mechanism that cells employ to tolerate replication stress, wherein stalled replication forks are remodelled into four-way junction structures, thereby facilitating replication restart while preventing DNA breakage. Several homologous recombination (HR) factors, including RAD51, RAD51-paralogs, and BRCA1/2, initially characterised for their roles in DSB repair, have also been shown to regulate replication stress by mediating fork reversal and restart of stalled replication forks. Dysregulated HR can lead to loss of heterozygosity, chromosomal rearrangements and tumorigenesis. Anti-recombinases, including RECQL5, RTEL1, PARI, FBH1, BLM and FIGNL1, counteract HR during DSB repair to limit the deleterious consequences of hyper-recombination. However, the mechanisms by which pro- and anti-recombinases regulate HR during replication and replication stress remain unclear. RECQL5 is an antirecombinase that dismantles RAD51 nucleoprotein filaments during the presynaptic stage of HR during DSB repair. In addition to its role in HR regulation, RECQL5 modulates transcription elongation and suppresses transcription-associated replication stress. However, the mechanisms by which RECQL5 coordinates these distinct processes remain poorly understood. Our findings uncover a novel role for RECQL5 in regulating RAD51-mediated fork reversal, distinct from its role in transcription elongation. SIRF analysis revealed that the RECQL5 is dynamically associated with active replication sites and gets further enriched at stalled forks. Loss of RECQL5 leads to genome-wide replication defects, including slow-moving replication forks, fork asymmetry, perturbed fork restart and genome instability. The genome-wide replication defects observed upon RECQL5 depletion were rescued by co-depletion of the fork remodelers HLTF/SMARCAL1/ZRANB3/FBH1, as well as by expression of HR-defective mutants of RAD51, suggesting that RECQL5 suppresses RAD51-mediated fork reversal. Moreover, RECQL5 depletion leads to persistent accumulation of RAD51 and RAD51 paralogs at the replication fork in response to replication stress. Loss of RECQL5 resulted in hyper-recombination at I-SceI-induced DSBs and at stalled replication forks, which was rescued by co-depletion of RAD51 paralogs, suggesting that RECQL5 counteracts HR factors at the stalled replication forks. The regulation of RAD51 at stalled fork sites by RECQL5 requires its binding to PCNA and RAD51, as well as its helicase activity. These results highlight the vital role played by RECQL5 in regulating HR at replicating sites to promote unrestrained DNA synthesis and genome stability. Furthermore, we elucidated that RECQL5 is upstream of RTEL1 in regulating RAD51 mediated fork reversal. We further found that RECQL5 mutants deficient in interaction with RNAPII, which resulted in impaired transcription elongation, remained proficient in regulating replication-associated functions. Notably, the RECQL5 mutant defective in RAD51 binding remained competent for transcription elongation. Collectively, these results establish that RECQL5 distinctly regulates RAD51-mediated fork remodeling during DNA replication and transcription elongation to promote error-free genome duplication.