Replication stress activates DNA polymerase alpha-associated Chk1.
Chk1 contributes to both intra-S and DNA damage checkpoint responses. Here, we show that depletion of DNA Polalpha and not Polepsilon or Poldelta by siRNA induces phosphorylation of Chk1 on Ser345, thus phenocopying antimetabolite exposure. Combinatorial ablation of DNA Polalpha and Chk1 causes an accumulation of gamma-H2A.X, a marker of double-strand DNA breaks, suggesting that activation of Chk1 in this context is essential for suppression of DNA damage. Co-depletion of DNA Polalpha with ATR yields similar phenotypes, suggesting that ATR and Chk1 are epistatic and required for maintenance of genomic integrity following replication stress. Significantly, Chk1 and DNA Polalpha can be co-immunoprecipated from native cell extracts. Moreover, following replication stress, Polalpha-associated Chk1 becomes rapidly phosphorylated on Ser345 in a TopBP1 and ATR-dependent manner. Hence, the ability to efficiently phosphorylate Chk1 in the context of DNA Polalpha complexes is correlated with suppression of DNA damage following replication stress. These findings identify DNA Polalpha as an important component of the signal transduction cascade that activates the intra-S checkpoint.
Taricani
Lorena
L
Discovery Research, Schering-Plough Biopharma, Palo Alto, California 94304-1104, USA.
Shanahan
Frances
F
Parry
David
D
D000963:Antimetabolites / Q000378:metabolism; D064007:Ataxia Telangiectasia Mutated Proteins; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002453:Cell Cycle / Q000502:physiology; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D000071877:Checkpoint Kinase 1; D004249:DNA Damage; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D051747:DNA-Activated Protein Kinase / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism
D000963:Antimetabolites; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D007527:Isoenzymes; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; C110513:TOPBP1 protein, human; D025521:Tumor Suppressor Proteins; D011494:Protein Kinases; C576324:ATM protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D004256:DNA Polymerase I