RESUMO
TIMELESS (TIM) in the fork protection complex acts as a scaffold of the replisome to prevent its uncoupling and ensure efficient DNA replication fork progression. Nevertheless, its underlying basis for coordinating leading and lagging strand synthesis to limit single-stranded DNA (ssDNA) exposure remains elusive. Here, we demonstrate that acute degradation of TIM at ongoing DNA replication forks induces the accumulation of ssDNA gaps stemming from defective Okazaki fragment (OF) processing. Cells devoid of TIM fail to support the poly(ADP-ribosyl)ation necessary for backing up the canonical OF processing mechanism mediated by LIG1 and FEN1. Consequently, recruitment of XRCC1, a known effector of PARP1-dependent single-strand break repair, to post-replicative ssDNA gaps behind replication forks is impaired. Physical disruption of the TIM-PARP1 complex phenocopies the rapid loss of TIM, indicating that the TIM-PARP1 interaction is critical for the activation of this compensatory pathway. Accordingly, combined deficiency of FEN1 and the TIM-PARP1 interaction leads to synergistic DNA damage and cytotoxicity. We propose that TIM is essential for the engagement of PARP1 to the replisome to coordinate lagging strand synthesis with replication fork progression. Our study identifies TIM as a synthetic lethal target of OF processing enzymes that can be exploited for cancer therapy.
Assuntos
Proteínas de Ciclo Celular , Replicação do DNA , DNA de Cadeia Simples , Peptídeos e Proteínas de Sinalização Intracelular , Poli(ADP-Ribose) Polimerase-1 , Humanos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , DNA/metabolismo , DNA/genética , DNA Ligase Dependente de ATP/metabolismo , DNA Ligase Dependente de ATP/genética , Reparo do DNA , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Endonucleases Flap/metabolismo , Endonucleases Flap/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/metabolismo , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genéticaRESUMO
Poly(ADP-ribosyl)ation (PARylation), catalyzed mainly by poly(ADP-ribose) polymerase (PARP)1, is a key posttranslational modification involved in DNA replication and repair. Here, we report that TIMELESS (TIM), an essential scaffold of the replisome, is PARylated, which is linked to its proteolysis. TIM PARylation requires recognition of auto-modified PARP1 via two poly(ADP-ribose)-binding motifs, which primes TIM for proteasome-dependent degradation. Cells expressing the PARylation-refractory TIM mutant or under PARP inhibition accumulate TIM at DNA replication forks, causing replication stress and hyper-resection of stalled forks. Mechanistically, aberrant engagement of TIM with the replicative helicase impedes RAD51 loading and protection of reversed forks. Accordingly, defective TIM degradation hypersensitizes BRCA2-deficient cells to replication damage. Our study defines TIM as a substrate of PARP1 and elucidates how the control of replisome remodeling by PARylation is linked to stalled fork protection. Therefore, we propose a mechanism of PARP inhibition that impinges on the DNA replication fork instability caused by defective TIM turnover.