Abstract
The Timeless-Tipin complex and Claspin are mediators of the ATR-dependent activation of Chk1 in the intra-S checkpoint response to stalled DNA replication forks. Tim-Tipin and Claspin also contribute to sister chromatid cohesion (SCC) in various organisms, likely through a replication-coupled process. Some models of the establishment of SCC posit that interactions between cohesin rings and replisomes could result in physiological replication stress requiring fork stabilization. The contributions of Timeless, Tipin, Claspin, Chk1 and ATR to SCC were investigated in genetically stable, human diploid fibroblast cell lines. Whereas Timeless, Tipin and Claspin showed similar contributions to UVC-induced activation of Chk1, siRNA-mediated knockdown of Timeless induced a 100-fold increase in sister chromatid discohesion, whereas the inductive effects of knocking down Tipin, Claspin and ATR were 4–20-fold. Knockdown of Chk1 did not significantly affect SCC. Consistent findings were obtained in two independently derived human diploid fibroblast lines and support a conclusion that SCC in human cells is strongly dependent on Timeless but independent of Chk1. Furthermore, the 10-fold difference in discohesion observed when depleting Timeless versus Tipin indicates that Timeless has a function in SCC that is independent of the Tim-Tipin complex, even though the abundance of Timeless is reduced when Tipin is targeted for depletion. A better understanding of how Timeless, Tipin and Claspin promote SCC will elucidate non-checkpoint functions of these proteins at DNA replication forks and inform models of the establishment of SCC.
Highlights
The ataxia telangiectasia-mutated and Rad3-related (ATR)dependent intra-S checkpoint is activated when excess replication protein A (RPA)-coated ssDNA is generated at replication forks that are stalled by DNA damage or by chemicals that inhibit DNA synthesis.[1]
The results indicate that the requirement for Timeless, Tipin and Claspin in sister chromatid cohesion (SCC) in human cells is independent of ataxia telangiectasia mutated and Rad3-related (ATR)-dependent intra-S checkpoint signaling
When NHF1-human telomerase reverse transcriptase (hTERT) were electroporated with small interfering RNA (siRNA) targeting Chk[1], Timeless, Tipin or Claspin, the targeted protein was reduced, but the levels of the other three proteins decreased by 40–50%
Summary
The ataxia telangiectasia-mutated and Rad3-related (ATR)dependent intra-S checkpoint is activated when excess replication protein A (RPA)-coated ssDNA is generated at replication forks that are stalled by DNA damage or by chemicals that inhibit DNA synthesis.[1]. Timeless and Tipin form a heterodimer and are components of the “replication fork protection complex” (RFPC), named for its role in stabilizing stalled DNA replication forks and protecting cells from genomic instability.[9,10] SiRNA-mediated reduction of Timeless, Tipin or Claspin proteins attenuates DNA damageinduced phosphorylation of Chk[1] and compromises ultraviolet (UV) light-induced activation of the intra-S checkpoint in HeLa cells.[3,6,8,11] Tim-Tipin and Claspin likely work together to mediate ATR activation of Chk[1] by Tipin-RPA recruitment of ClaspinChk[1] through a Tipin-Claspin interaction.[8] TimTipin and Claspin orthologs/analogs associate with chromatin, interact with replisome components and appear to travel with replication forks in the absence of exogenously applied DNA damage,[9,10,12,13,14,15,16] and DNA synthesis is compromised when human cells are depleted of Timeless, Tipin or Claspin.[3,6,17]
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