Sister chromatid exchanges induced by perturbed replication can form independently of BRCA1, BRCA2 and RAD51

Abstract

Sister chromatid exchanges (SCEs) are products of joint DNA molecule resolution, and are considered to form through homologous recombination (HR). Indeed, SCE induction upon irradiation requires the canonical HR factors BRCA1, BRCA2 and RAD51. In contrast, replication-blocking agents, including PARP inhibitors, induce SCEs independently of BRCA1, BRCA2 and RAD51. PARP inhibitor-induced SCEs are enriched at difficult-to-replicate genomic regions, including common fragile sites (CFSs). PARP inhibitor-induced replication lesions are transmitted into mitosis, suggesting that SCEs can originate from mitotic processing of under-replicated DNA. Proteomics analysis reveals mitotic recruitment of DNA polymerase theta (POLQ) to synthetic DNA ends. POLQ inactivation results in reduced SCE numbers and severe chromosome fragmentation upon PARP inhibition in HR-deficient cells. Accordingly, analysis of CFSs in cancer genomes reveals frequent allelic deletions, flanked by signatures of POLQ-mediated repair. Combined, we show PARP inhibition generates under-replicated DNA, which is processed into SCEs during mitosis, independently of canonical HR factors.

RHINO directs MMEJ to repair DNA breaks in mitosis

“…Recently, a tethering complex comprising MDC1-CIP2A-TOPBP1 was reported to hold broken mitotic DNA ends together until cells progress into the next G 1 ( 33 , 47 , 48 ). Multiple studies implicated Polθ activity in repairing DNA damage in mitosis ( 27 – 30 ). An investigation using Xenopus egg extract showed that entry into mitosis before the completion of DNA replication leads to complex rearrangements driven by Polθ ( 29 ).…”

“…In human and mouse cells, MMEJ acts with NHEJ to promote the random integration of foreign DNA into the genome and repair CRISPR-Cas9–induced breaks at particular loci ( 24 – 26 ). Furthermore, recent evidence suggests that MMEJ plays a role in DSBs during mitosis, where HR and NHEJ are attenuated ( 27 – 30 ).…”

Targeting the BRCA1/2 deficient cancer with PARP inhibitors: Clinical outcomes and mechanistic insights

“…Similarly, using the spectral karyotyping technique, Lavoie and colleagues did not observe elevated expression of fragility in PARP1-null mouse cells either ( Lavoie et al, 2005 ). However, using the recently developed Strand-seq technology, which can map genome-wide sister chromatid exchange (SCE) via single-cell sequencing, Heijink and colleagues showed that PARPi-induced SCEs frequently take place at many CFSs ( Heijink et al, 2022 ). Notably, they demonstrated that these SCE events are independent of BRCA1 and BRCA2, suggesting that PARP1 likely acts in a parallel pathway facilitating the repair of replication blockage at the CFSs.…”

Polθ is phosphorylated by Polo-like kinase 1 (PLK1) to enable repair of DNA double strand breaks in mitosis

“…In mitosis, while it is well accepted that HR and NHEJ repair activities are inhibited, the activity of alternative pathways remains unaddressed. While there has not been direct evidence of DSB repair in mitosis, recent data suggests that alt-EJ 28,29 and Polθ could be active in mitosis [30][31][32] took advantage of recent advances in genome editing, allowing the induction of DSBs within minutes after Cas9 protein transfection. Nocodazole-arrested cells were transfected with a Cas9/gRNA complex to induce DSB formation at two different sites in the AAVS1 safe locus, whose mutagenic repair would destroy a nearby HphI restriction site.…”

“…In mitosis, while it is well accepted that HR and NHEJ repair activities are inhibited, the activity of alternative pathways remains unaddressed. While there has not been direct evidence of DSB repair in mitosis, recent data suggests that alt-EJ 28,29 and Polθ could be active in mitosis [30][31][32] . To detect DSB repair in mitosis, we took advantage of recent advances in genome editing, allowing the induction of DSBs within minutes after Cas9 protein transfection.…”