PIF1 helicase promotes break‐induced replication in mammalian cells

Abstract

Break‐induced replication (BIR) is a specialized homologous‐recombination pathway for DNA double‐strand break (DSB) repair, which often induces genome instability. In this study, we establish EGFP‐based recombination reporters to systematically study BIR in mammalian cells and demonstrate an important role of human PIF1 helicase in promoting BIR. We show that at endonuclease cleavage sites, PIF1‐dependent BIR is used for homology‐initiated recombination requiring long track DNA synthesis, but not short track gene conversion (STGC). We also show that structure formation‐prone AT‐rich DNA sequences derived from common fragile sites (CFS‐ATs) induce BIR upon replication stress and oncogenic stress, and PCNA‐dependent loading of PIF1 onto collapsed/broken forks is critical for BIR activation. At broken replication forks, even STGC‐mediated repair of double‐ended DSBs depends on POLD3 and PIF1, revealing an unexpected mechanism of BIR activation upon replication stress that differs from the conventional BIR activation model requiring DSB end sensing at endonuclease‐generated breaks. Furthermore, loss of PIF1 is synthetically lethal with loss of FANCM, which is involved in protecting CFS‐ATs. The breast cancer‐associated PIF1 mutant L319P is defective in BIR, suggesting a direct link of BIR to oncogenic processes.

Homologous Recombination Subpathways: A Tangle to Resolve

“…demonstrated recently, the loss of the BIR factor PIF-1 can be exploited for selective killing of cells made to rely on this HR subpathway by the concurrent deletion of FANCM, revealing a new synthetic lethality relationship and an approach to target PIF-1 mutant cancer cells (Li et al, 2021). Also, it is known that the HR factor ATRX is defective in a variety of tumors that are commonly using the alternative lengthening of telomeres (ALT) mechanism of telomere maintenance (representing around 10-15% of all cancers; Dilley and Greenberg, 2015).…”

Translesion Synthesis or Repair by Specialized DNA Polymerases Limits Excessive Genomic Instability upon Replication Stress

“…In fact, unlike other DSBs, DSBs at collapsed forks are single ended, with no second end available for classical HR repair. These breaks can be processed by a replication-coupled repair named break-induced replication (BIR), a conservative DNA synthesis mechanism described as an HR-based repair pathway for one-ended DNA DSBs, which has been studied extensively in budding yeast [99] and more recently in mammalian cells [100]. BIR initiates at the broken forks, proceeds through the invasion by the broken chromosome of its homologous duplex, and moves as a D-loop along the length of the chromosome.…”

Srs2 and Pif1 as Model Systems for Understanding Sf1a and Sf1b Helicase Structure and Function

“…Finally, recent work has shown that Pif1 is also necessary for BIR in human cell lines [ 138 ]. Importantly, this study demonstrated that the breast cancer-associated Pif1 mutant L319P, which resides within the Pif1 family signature motif, was defective for BIR [ 138 ].…”

“…Finally, recent work has shown that Pif1 is also necessary for BIR in human cell lines [ 138 ]. Importantly, this study demonstrated that the breast cancer-associated Pif1 mutant L319P, which resides within the Pif1 family signature motif, was defective for BIR [ 138 ]. This work provides crucial evidence suggesting that not only is the role of Pif1 in BIR is likely to be broadly conserved among eukaryotes, but also providing a direct indication that BIR (and Pif1) may play roles in human genome integrity and cancers.…”