title: Separation of intra-S checkpoint protein contributions to DNA replication fork protection and genomic stability in normal human fibroblasts.
abstract: The ATR-dependent intra-S checkpoint protects DNA replication forks undergoing replication stress. The checkpoint is enforced by ATR-dependent phosphorylation of CHK1, which are mediated by the TIMELESS-TIPIN complex and CLASPIN. Although loss of checkpoint proteins is associated with spontaneous chromosomal instability, few studies have examined the contribution of these proteins to unchallenged DNA metabolism in human cells that have not undergone carcinogenesis or crisis. Furthermore, the TIMELESS-TIPIN complex and CLASPIN may promote replication fork protection independently of CHK1 activation. Normal human fibroblasts (NHF) were depleted of ATR, CHK1, TIMELESS, TIPIN or CLASPIN and chromosomal aberrations, DNA synthesis, activation of the DNA damage response (DDR) and clonogenic survival were evaluated. This work demonstrates in NHF lines from two individuals that ATR and CHK1 promote chromosomal stability by different mechanisms that depletion of CHK1 produces phenotypes that resemble more closely the depletion of TIPIN or CLASPIN than the depletion of ATR, and that TIMELESS has a distinct contribution to suppression of chromosomal instability that is independent of its heterodimeric partner, TIPIN. Therefore, ATR, CHK1, TIMELESS-TIPIN and CLASPIN have functions for preservation of intrinsic chromosomal stability that is separate from their cooperation for activation of the intra-S checkpoint response to experimentally induced replication stress. These data reveal a complex and coordinated program of genome maintenance enforced by proteins known for their intra-S checkpoint function.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. stephanie.smith-roe@nih.gov
authors.forename: Stephanie L
authors.initials: SL
authors.lastname: Smith-Roe
authors.forename: Shivani S
authors.initials: SS
authors.lastname: Patel
authors.forename: Yingchun
authors.initials: Y
authors.lastname: Zhou
authors.forename: Dennis A
authors.initials: DA
authors.lastname: Simpson
authors.forename: Shangbang
authors.initials: S
authors.lastname: Rao
authors.forename: Joseph G
authors.initials: JG
authors.lastname: Ibrahim
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 / Q000172:deficiency / Q000378:metabolism; D064007:Ataxia Telangiectasia Mutated Proteins; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000172:deficiency / Q000378:metabolism*; D000071877:Checkpoint Kinase 1; D002869:Chromosome Aberrations; D004261:DNA Replication / Q000502:physiology*; D004268:DNA-Binding Proteins; D005347:Fibroblasts; D005434:Flow Cytometry; D042822:Genomic Instability / Q000502:physiology*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000172:deficiency / Q000378:metabolism*; D016013:Likelihood Functions; D009687:Nuclear Proteins / Q000172:deficiency / Q000378:metabolism; D011494:Protein Kinases / Q000172:deficiency / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000172:deficiency / Q000378:metabolism*; D059807:S Phase Cell Cycle Checkpoints / Q000502:physiology*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C488158:CLSPN protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; C509769:TIMELESS protein, human; C516227:Tipin protein, human; D011494:Protein Kinases; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases