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 Pol alpha and not Pol epsilon or Pol delta by siRNA induces phosphorylation of Chk1 on Ser345, thus phenocopying antimetabolite exposure. Combinatorial ablation of DNA Pol alpha 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 Pol alpha 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 Pol alpha can be co-immunoprecipated from native cell extracts. Moreover, following replication stress, Pol alpha-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 Pol alpha complexes is correlated with suppression of DNA damage following replication stress. These findings identify DNA Pola as an important component of the signal transduction cascade that activates the intra-S checkpoint.