Tim-Tipin dysfunction creates an indispensible reliance on the ATR-Chk1 pathway for continued DNA synthesis.
The Tim (Timeless)-Tipin complex has been proposed to maintain genome stability by facilitating ATR-mediated Chk1 activation. However, as a replisome component, Tim-Tipin has also been suggested to couple DNA unwinding to synthesis, an activity expected to suppress single-stranded DNA (ssDNA) accumulation and limit ATR-Chk1 pathway engagement. We now demonstrate that Tim-Tipin depletion is sufficient to increase ssDNA accumulation at replication forks and stimulate ATR activity during otherwise unperturbed DNA replication. Notably, suppression of the ATR-Chk1 pathway in Tim-Tipin-deficient cells completely abrogates nucleotide incorporation in S phase, indicating that the ATR-dependent response to Tim-Tipin depletion is indispensible for continued DNA synthesis. Replication failure in ATR/Tim-deficient cells is strongly associated with synergistic increases in H2AX phosphorylation and DNA double-strand breaks, suggesting that ATR pathway activation preserves fork stability in instances of Tim-Tipin dysfunction. Together, these experiments indicate that the Tim-Tipin complex stabilizes replication forks both by preventing the accumulation of ssDNA upstream of ATR-Chk1 function and by facilitating phosphorylation of Chk1 by ATR.
Smith
Kevin D
KD
Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
Fu
Michael A
MA
Brown
Eric J
EJ
D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D002460:Cell Line; D000071877:Checkpoint Kinase 1; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000096:biosynthesis* / Q000235:genetics; D004268:DNA-Binding Proteins; D004789:Enzyme Activation; D005822:Genetic Vectors; D007150:Immunohistochemistry; D047908:Intracellular Signaling Peptides and Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D008957:Models, Genetic; D041681:NIH 3T3 Cells; D009687:Nuclear Proteins / Q000737:chemistry / Q000172:deficiency / Q000378:metabolism* / Q000502:physiology; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D034741:RNA, Small Interfering / Q000378:metabolism
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; C509770:Timeless protein, mouse; C518120:Tipin protein, mouse; D011494:Protein Kinases; C576897:Atr protein, mouse; D064007:Ataxia Telangiectasia Mutated Proteins; D000071877:Checkpoint Kinase 1; C000605869:Chek1 protein, mouse; D017346:Protein Serine-Threonine Kinases