Poly(ADP-ribosyl)ation of TIMELESS limits DNA replication stress and promotes stalled fork protection.

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. [Display omitted] • TIMELESS (TIM) and PARP1 interact at DNA replication forks • TIM recognizes auto-PARylated PARP1 by two conserved PAR-binding motifs (PBMs) • PBM-dependent TIM PARylation primes TIM for proteasomal degradation • Aberrant TIM accumulation causes replication stress and impairs stalled fork protection Rageul et al. report that TIMELESS harbors poly(ADP-ribose) (PAR)-binding motifs, which are essential for the PARylation of TIMELESS by PARP1. Defects in this modification prevent proteasomal degradation of TIMELESS and cause DNA replication fork instability. The proteotoxic stress caused by dysregulated TIMELESS turnover underlies a mechanism of PARP inhibitor action. [ABSTRACT FROM AUTHOR]