Abstract
Eukaryotic replication origins are licensed by the loading of the replicative DNA helicase, Mcm2-7, in inactive double hexameric form around DNA. Subsequent origin activation is under control of multiple protein kinases that either promote or inhibit origin activation, which is important for genome maintenance. Using the reconstituted budding yeast DNA replication system, we find that the flexible N-terminal extension (NTE) of Mcm2 promotes the stable recruitment of Dbf4-dependent kinase (DDK) to Mcm2-7 double hexamers, which in turn promotes DDK phosphorylation of Mcm4 and -6 and subsequent origin activation. Conversely, we demonstrate that the checkpoint kinase, Rad53, inhibits DDK binding to Mcm2-7 double hexamers. Unexpectedly, this function is not dependent on Rad53 kinase activity, suggesting steric inhibition of DDK by activated Rad53. These findings identify critical determinants of the origin activation reaction and uncover a novel mechanism for checkpoint-dependent origin inhibition.
Highlights
To ensure the timely, accurate, and complete duplication of their genomes prior to cell division, eukaryotic cells initiate DNA replication at many replication origins distributed along the length of each chromosome
Helicase activation is controlled by two cell cycle-regulated protein kinases, Dbf4-dependent kinase (DDK) and cyclin-dependent kinase (CDK), which act in conjunction with a defined set of co-factors, comprising Sld3·7, Sld2, Dpb11, Pol e, and Mcm10, in addition to GINS and Cdc45, to mediate CMG assembly (Douglas et al, 2018)
As nuclear import is irrelevant for DNA replication with purified proteins, and chromatin can be omitted from DNA replication reactions in vitro, we asked whether the Mcm2 N-terminal tail possesses a basic DNA replication function
Summary
Accurate, and complete duplication of their genomes prior to cell division, eukaryotic cells initiate DNA replication at many replication origins distributed along the length of each chromosome. Mcm DHs are catalytically inactive and require the regulated association of the essential helicase co-factors Cdc and GINS to form two active replicative DNA helicase complexes, termed CMG (Cdc45-MCM-GINS), which encircle single-stranded DNA (ssDNA) during unwinding (Bell and Labib, 2016). The replication of chromosomes from multiple replication origins necessitates strict control mechanisms that prevent origins from re-firing within one cell cycle in order to maintain genome stability Such re-replication control is achieved by a two-step mechanism that temporally separates helicase loading in late M and G1 phase from helicase activation in S phase (Bell and Labib, 2016). Helicase activation is controlled by two cell cycle-regulated protein kinases, Dbf4-dependent kinase (DDK) and cyclin-dependent kinase (CDK), which act in conjunction with a defined set of co-factors, comprising Sld3·7, Sld, Dpb, Pol e, and Mcm, in addition to GINS and Cdc, to mediate CMG assembly (Douglas et al, 2018). If these mutations allow Sld recruitment in the absence of Mcm phosphorylation, or if they bypass the requirement for Sld altogether
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