title: Timeless functions independently of the Tim-Tipin complex to promote sister chromatid cohesion in normal human fibroblasts.
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.
authors.affiliation: University of North Carolina at Chapel Hill, Chapel Hill, NC USA. smithroe@email.unc.edu
authors.forename: Stephanie L
authors.initials: SL
authors.lastname: Smith-Roe
authors.forename: Shivani S
authors.initials: SS
authors.lastname: Patel
authors.forename: Dennis A
authors.initials: DA
authors.lastname: Simpson
authors.forename: Ying Chun
authors.initials: YC
authors.lastname: Zhou
authors.forename: Shangbang
authors.initials: S
authors.lastname: Rao
authors.forename: Joseph G
authors.initials: JG
authors.lastname: Ibrahim
authors.forename: Kathleen A
authors.initials: KA
authors.lastname: Kaiser-Rogers
authors.forename: Marila
authors.initials: M
authors.lastname: Cordeiro-Stone
authors.forename: William K
authors.initials: WK
authors.lastname: Kaufmann
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D064007:Ataxia Telangiectasia Mutated Proteins; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000071877:Checkpoint Kinase 1; D002842:Chromatids / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D004261:DNA Replication; D004268:DNA-Binding Proteins; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008677:Metaphase; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D014466:Ultraviolet Rays; D000097722:Cohesins
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D064007:Ataxia Telangiectasia Mutated Proteins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D000071877:Checkpoint Kinase 1; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D034741:RNA, Small Interfering; D000097722:Cohesins; C488158:CLSPN protein, human; C509769:TIMELESS protein, human; C516227:Tipin protein, human; C576597:ATR protein, human; C000605867:CHEK1 protein, human