Poly(ADP-ribose) binding to Chk1 at stalled replication forks is required for S-phase checkpoint activation

Abstract

Damaged replication forks activate poly(ADP-ribose) polymerase 1 (PARP1), which catalyses poly(ADP-ribose) (PAR) formation; however, how PARP1 or poly(ADP-ribosyl)ation is involved in the S-phase checkpoint is unknown. Here we show that PAR, supplied by PARP1, interacts with Chk1 via a novel PAR-binding regulatory (PbR) motif in Chk1, independent of ATR and its activity. iPOND studies reveal that Chk1 associates readily with the unperturbed replication fork and that PAR is required for efficient retention of Chk1 and phosphorylated Chk1 at the fork. A PbR mutation, which disrupts PAR binding, but not the interaction with its partners Claspin or BRCA1, impairs Chk1 and the S-phase checkpoint activation, and mirrors Chk1 knockdown-induced hypersensitivity to fork poisoning. We find that long chains, but not short chains, of PAR stimulate Chk1 kinase activity. Collectively, we disclose a previously unrecognized mechanism of the S-phase checkpoint by PAR metabolism that modulates Chk1 activity at the replication fork.

A balanced pyrimidine pool is required for optimal Chk1 activation to prevent ultrafine anaphase bridge formation

“…2A; Fig. S2A), but PARP-1-depleted cells displayed higher levels of DNA synthesis than PARP-1-expressing cells, as previously reported (Min et al, 2013) (Fig. S2A).…”

“…Chk1 was found to be less abundant in the chromatin-bound fraction of PARP-1-deficient cells than in that of control cells. We thus confirmed that PARP-1 defects decrease the stability of Chk1 on DNA (Min et al, 2013) (Fig. S1A).…”

“…Chk1 phosphorylation levels were lower in PARP-1-deficient cells than in control cells (Fig. S1B), confirming that PARP-1 contributes to optimal Chk1 activation (Min et al, 2013). In CDA-deficient cells, Chk1 phosphorylation levels were 34% lower than those in CDA-expressing cells after exposure to UVC (Fig.…”

“…The interaction of Chk1 with PAR is required for its efficient retention on DNA, and, thus, for its optimal activation (Min et al, 2013). We hypothesized that the lower levels of PARP-1 activity in CDA-deficient cells might decrease the stability of Chk1 on DNA and its activation.…”

Poly(ADP-Ribosyl) Glycohydrolase Prevents the Accumulation of Unusual Replication Structures during Unperturbed S Phase

“…It should be noted, however, that other experimental conditions recently reported to induce similar levels of reversed replication forks have not been associated with DDR activation (11,(32)(33)(34), excluding the possibility that fork reversal per se is sufficient to induce checkpoint activation and chromatin recruitment of DSB repair factors. Checkpoint activation upon PAR accumulation may directly reflect the recently reported physical association of PAR with checkpoint factors, such as CHK1 (35). Alternatively, the persistence of reversed forks upon impairment of PAR degradation may lead to recruitment of cellular factors usually recruited at DSB (53BP1 and RAD51), either before or after nucleolytic processing of the stalled reversed forks (Fig.…”