Novel role of DONSON in CMG helicase assembly during vertebrate DNA replication initiation

Abstract

CMG (Cdc45‐MCM‐GINS) helicase assembly at the replication origin is the culmination of eukaryotic DNA replication initiation. This process can be reconstructed in vitro using defined factors in Saccharomyces cerevisiae ; however, in vertebrates, origin‐dependent CMG formation has not yet been achieved partly due to the lack of a complete set of known initiator proteins. Since a microcephaly gene product, DONSON, was reported to remodel the CMG helicase under replication stress, we analyzed its role in DNA replication using a Xenopus cell‐free system. We found that DONSON was essential for the replisome assembly. In vertebrates, DONSON physically interacted with GINS and Polε via its conserved N‐terminal PGY and NPF motifs, and the DONSON‐GINS interaction contributed to the replisome assembly. DONSON's chromatin association during replication initiation required the pre‐replicative complex, TopBP1, and kinase activities of S‐CDK and DDK. Both S‐CDK and DDK required DONSON to trigger replication initiation. Moreover, human DONSON could substitute for the Xenopus protein in a cell‐free system. These findings indicate that vertebrate DONSON is a novel initiator protein essential for CMG helicase assembly. image Vertebrate DONSON is involved in replication fork stability as a component of the replisome complex. This study reveals that DONSON also functions as an initiator protein to promote CMG (Cdc45‐MCM‐GINS) helicase assembly through the interaction with GINS in the initiation of DNA replication. DONSON is required for CMG helicase assembly during DNA replication initiation. DONSON physically interacts with Cdc45, GINS, and Polε via its N‐terminal disordered region. The DONSON‐GINS interaction contributes to CMG helicase assembly. DONSON's chromatin association during replication initiation requires the pre‐replicative complex, TopBP1, and kinase activities of S‐CDK and DDK. S‐CDK and DDK require DONSON function to trigger replication initiation.