Abstract
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.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Ataxia Telangiectasia Mutated Proteins
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Carrier Proteins / chemistry
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Carrier Proteins / metabolism*
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Carrier Proteins / physiology
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism*
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Cell Cycle Proteins / physiology
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Cell Line
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Checkpoint Kinase 1
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DNA Replication
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DNA, Single-Stranded / biosynthesis*
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DNA, Single-Stranded / genetics
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DNA-Binding Proteins
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Enzyme Activation
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Genetic Vectors
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Immunohistochemistry
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Intracellular Signaling Peptides and Proteins / deficiency
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Intracellular Signaling Peptides and Proteins / genetics
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Intracellular Signaling Peptides and Proteins / metabolism*
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Intracellular Signaling Peptides and Proteins / physiology
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Lentivirus / genetics
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Mice
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Models, Genetic
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NIH 3T3 Cells
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Nuclear Proteins / chemistry
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Nuclear Proteins / deficiency
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Nuclear Proteins / metabolism*
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Nuclear Proteins / physiology
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Phosphorylation
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Protein Kinases / genetics
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Protein Kinases / metabolism*
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Protein Kinases / physiology
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Protein Serine-Threonine Kinases / genetics
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Protein Serine-Threonine Kinases / metabolism*
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Protein Serine-Threonine Kinases / physiology
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RNA, Small Interfering / metabolism
Substances
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Carrier Proteins
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Cell Cycle Proteins
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DNA, Single-Stranded
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DNA-Binding Proteins
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Intracellular Signaling Peptides and Proteins
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Nuclear Proteins
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RNA, Small Interfering
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Timeless protein, mouse
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Tipin protein, mouse
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Protein Kinases
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Atr protein, mouse
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Ataxia Telangiectasia Mutated Proteins
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Checkpoint Kinase 1
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Chek1 protein, mouse
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Protein Serine-Threonine Kinases