Phosphorylation of Mcm4 at specific sites by cyclin-dependent kinase leadsto loss of Mcm4,6,7 helicase activity

Mcm proteins that play an essential role in eukaryotic DNA replication are phosphorylated in vivo, and cyclin-dependent protein kinase is at least in part responsible for the phosphorylation of Mcm4. Our group reported that t he DNA helicase activity of Mcm4,6,7 complex, which may be involved in init iation of DNA replication, is inhibited following phosphorylation by Cdk2/c yclin A in vitro. Here, we further examined the interplay between mouse Mcm 4,6,7 complex and cyclin-dependent kinases and determined the sites require d for the phosphorylation of Mcm4. Six Ser and Thr residues, in all, were r equired for the phosphorylation. Inhibition of Mcm4,6,7 helicase activity b y Cdk2/cyclin A was largely relieved by introducing mutations in these resi dues Mcm4. Anti-phosphothreonine antibodies raised against one of these sit es reacted with Mcm4 prepared fro HeLa cells at mitotic phase but did not b ind to those at G(1) and G(1)/S, suggesting that this site is mainly phosph orylated in the mitotic phase. Mcm4,6,7 complex purified from HeLa cells at the mitotic phase exhibited a low level of DNA helicase activity, compared with the complexes prepared from cells at other phases. These results sugg est that phosphorylation of Mcm4 at specific sites leads to loss of Mcm4,6, 7 DNA helicase activity.