A conserved phosphorylation mechanism for regulating the interaction between the CMG replicative helicase and its forked DNA substrate.

Abstract

The CMG helicase is a crucial enzyme complex that plays a vital role in the replication of genomic DNA in eukaryotes. Besides unwinding the DNA template and coordinating the replisome's structure, it is also a key target for signaling pathways that regulate the replication process. We show that a specific serine/threonine residue in the MCM3 subunit of CMG, which has been previously linked to phosphorylation-dependent control mechanisms of genomic DNA replication in human cells, is a conserved phosphorylation site for Chk1 and potentially other protein kinases. This suggests a conserved regulatory mechanism associated with it in metazoans and several other eukaryotes, including budding yeast. Our in vitro analysis links this mechanism directly to the modulation of the CMG helicase activity by impacting its interactions with the forked DNA substrate. Further supporting its conserved role in regulation, we found that phosphomimetic substitution with aspartic acid and alanine knock-out of this conserved residue lead to opposite phenotypic defects in the growth of budding yeast cells. These findings outline a candidate conserved phosphorylation pathway for regulating genomic DNA replication in eukaryotes, which adjusts the interactions between the replicative helicase complex and its DNA substrate according to the specific needs of various physiological conditions.