Mitotic kinase regulation of DNA replication forks

While DNA replication forks initiate in S phase, they do not necessarily complete and terminate prior to cell entry into mitosis. How mitotic proteins regulate leftover replication forks is not well-understood. Using reconstituted DNA replication forks with purified proteins, we show that the budding yeast mitotic kinases Clb2-CDK (M-CDK) and Cdc5 (Plk1 homolog) phosphorylate and regulate several replication elongation proteins. Mrc1 phosphorylation by both kinases results in slower replication, and Polα phosphorylation by M-CDK results in less lagging strand initiation. We further show that a phospho-resistant mutant of Polα bypasses M-CDK inhibition of Polα activity in reconstituted replication reactions. Yeast cells expressing the phospho-resistant mutant exhibit faster cell cycle progression revealing a potential negative feedback mechanism between DNA replication forks and mitotic progression.