The Chk1-mediated S-phase checkpoint targets initiation factor Cdc45 via a Cdc25A/Cdk2-independent mechanism.
DNA damage induced by the carcinogen benzo[a]pyrene dihydrodiol epoxide (BPDE) induces a Chk1-dependent S-phase checkpoint. Here, we have investigated the molecular basis of BPDE-induced S-phase arrest. Chk1-dependent inhibition of DNA synthesis in BPDE-treated cells occurred without detectable changes in Cdc25A levels, Cdk2 activity, or Cdc7/Dbf4 interaction. Overexpression studies showed that Cdc25A, cyclin A/Cdk2, and Cdc7/Dbf4 were not rate-limiting for DNA synthesis when the BPDE-induced S-phase checkpoint was active. To investigate other potential targets of the S-phase checkpoint, we tested the effects of BPDE on the chromatin association of DNA replication factors. The levels of chromatin-associated Cdc45 (but not soluble Cdc45) were reduced concomitantly with BPDE-induced Chk1 activation and inhibition of DNA synthesis. The chromatin association of Mcm7, Mcm10, and proliferating cell nuclear antigen was unaffected by BPDE treatment. However, the association between Mcm7 and Cdc45 in the chromatin fraction was inhibited in BPDE-treated cells. Chromatin immunoprecipitation analyses demonstrated reduced association of Cdc45 with the beta-globin origin of replication in BPDE-treated cells. The inhibitory effects of BPDE on DNA synthesis, Cdc45/Mcm7 associations, and interactions between Cdc45 and the beta-globin locus were abrogated by the Chk1 inhibitor UCN-01. Taken together, our results show that the association between Cdc45 and Mcm7 at origins of replication is negatively regulated by Chk1 in a Cdk2-independent manner. Therefore, Cdc45 is likely to be an important target of the Chk1-mediated S-phase checkpoint.
Liu
Peijun
P
Department of Genetics and Genomics, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Barkley
Laura R
LR
Day
Tovah
T
Bi
Xiaohui
X
Slater
Damien M
DM
Alexandrow
Mark G
MG
Nasheuer
Heinz-Peter
HP
Vaziri
Cyrus
C
D018797:Cell Cycle Proteins / Q000096:biosynthesis* / Q000378:metabolism; D000071877:Checkpoint Kinase 1; D002843:Chromatin / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D064168:Minichromosome Maintenance Complex Component 7; D064110:Minichromosome Maintenance Proteins; D009687:Nuclear Proteins / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011494:Protein Kinases / Q000502:physiology*; D016196:S Phase; D019311:Staurosporine / Q000031:analogs & derivatives / Q000494:pharmacology; D020687:cdc25 Phosphatases / Q000096:biosynthesis*
C113571:CDC45 protein, human; D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; C489613:MCM10 protein, human; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; C054852:7-hydroxystaurosporine; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D051357:Cyclin-Dependent Kinase 2; C517004:CDC25A protein, human; D020687:cdc25 Phosphatases; C101624:MCM7 protein, human; D064168:Minichromosome Maintenance Complex Component 7; D064110:Minichromosome Maintenance Proteins; D019311:Staurosporine