Kinase-independent function of checkpoint kinase 1 (Chk1) in the replication of damaged DNA

Abstract

The checkpoint kinases Chk1 and ATR are broadly known for their role in the response to the accumulation of damaged DNA. Because Chk1 activation requires its phosphorylation by ATR, it is expected that ATR or Chk1 down-regulation should cause similar alterations in the signals triggered by DNA lesions. Intriguingly, we found that Chk1, but not ATR, promotes the progression of replication forks after UV irradiation. Strikingly, this role of Chk1 is independent of its kinase-domain and of its partnership with Claspin. Instead, we demonstrate that the ability of Chk1 to promote replication fork progression on damaged DNA templates relies on its recently identified proliferating cell nuclear antigen-interacting motif, which is required for its release from chromatin after DNA damage. Also supporting the importance of Chk1 release, a histone H2B-Chk1 chimera, which is permanently immobilized in chromatin, is unable to promote the replication of damaged DNA. Moreover, inefficient chromatin dissociation of Chk1 impairs the efficient recruitment of the specialized DNA polymerase η (pol η) to replication-associated foci after UV. Given the critical role of pol η during translesion DNA synthesis (TLS), these findings unveil an unforeseen facet of the regulation by Chk1 of DNA replication. This kinase-independent role of Chk1 is exclusively associated to the maintenance of active replication forks after UV irradiation in a manner in which Chk1 release prompts TLS to avoid replication stalling. T he checkpoint kinases ATR and Chk1 are central factors in the DNA damage response (1). During the S phase checkpoint, ATR is activated at single-stranded DNA (ssDNA) and this event, in turn, activates the effector kinase Chk1. Although ATR remains associated with the DNA, activated Chk1 rapidly spreads throughout the whole nucleus. Within the nucleoplasm, Chk1 delays the progression through S phase via phosphorylation of key target genes (2, 3).Several lines of evidence suggest that the activities of ATR and Chk1 are also required for proper unperturbed S phase progression. In fact, ATR or Chk1 loss leads to embryonic lethality (4-7), and Chk1 heterozygosity is associated with multiple defects, including a miscoordinated cell cycle and increased apoptosis (8).A contribution of Chk1 to replication fork stability during unperturbed DNA replication was identified and characterized in detail (9-11). Chk1 activity promotes the maintenance of global replication rates by regulating origin firing. In line with these observations, the monoallelic expression of the mutant Chk1 S317A, which is not phosphorylated by ATR, impairs fork elongation (12). Together, these results reveal an unambiguous role of the Chk1 kinase during unperturbed DNA replication. Intriguingly, recent reports described a kinase-independent effect of Chk1 on DNA replication-associated events. Scorah and colleagues discovered a proliferating cell nuclear antigen (PCNA) binding motif of Chk1 (Chk1_TRFF motif) required for the efficient dissociation of Chk1 from chromatin a...

Separation of intra-S checkpoint protein contributions to DNA replication fork protection and genomic stability in normal human fibroblasts

“…CHK1 can associate with chromatin in the absence of applied replication stress and independently of ATR, TOPBP1, HUS1, NBS1 or CLASPIN, [54][55][56] and kinase-independent functions for CHK1 have been reported for replication of damaged DNA via interactions with PCNA. 40,57 The activation of the DDR in CHK1-depleted cells and accumulation in G 2 may indicate that sufficient activation of the G 2 checkpoint took place for successful repair of damaged DNA in cells that were able to complete S phase. However, some CHK1-depleted cells may have had such a high level of DNA damage To provide an overall summary and comparison of the results, an unsupervised, hierarchical heat map was created to cluster the various experimental endpoints obtained from NHF1-hTERT (Fig.…”

Combination Drug Scheduling Defines a “Window of Opportunity” for Chemopotentiation of Gemcitabine by an Orally Bioavailable, Selective ChK1 Inhibitor, GNE-900

“…Although there has been extensive literature on the role of ChK1 in S-and G 2 -M checkpoints, many of these earlier studies have been conducted with reagents that can invoke alternate mechanisms of chemopotentiation. For example, siRNA depletion removes the scaffolding function of ChK1 at replication forks (38), AZD-7762 inhibits both ChK1 and ChK2 (39,40), whereas UCN-01, a tool compound frequently used to inhibit ChK1, has poor overall kinase selectivity (41).…”

Regulation of the specialized DNA polymerase eta: Revisiting the biological relevance of its PCNA‐ and ubiquitin‐binding motifs

“…In the presence of DNA lesions, the accumulation of polh in replication foci may favor polh in this competition by increasing locally its concentration. In parallel, a growing body of evidence suggests that proteins bearing PIP-boxes are degraded after UVC to clear PCNA and allow polh focus formation [Soria et al, 2008;Speroni et al, 2012].…”