Abstract
Assembly of the Cdc45-Mcm2-7-GINS (CMG) replicative helicase complex must be regulated to ensure that DNA unwinding is coupled with DNA synthesis. Sld2 is required for the initiation of DNA replication in budding yeast. We identified a mutant of Sld2, Sld2-m1,4, that is specifically defective in Mcm2-7 binding. When this sld2-m1,4 mutant is expressed, cells exhibit severe inhibition of DNA replication. Furthermore, the CMG complex assembles prematurely in G1 in mutant cells, but not wild-type cells. These data suggest that Sld2 binding to Mcm2-7 is essential to block the inappropriate formation of a CMG helicase complex in G1. We also study a mutant of Sld2 that is defective in binding DNA, sld2-DNA, and find that sld2-DNA cells exhibit no GINS-Mcm2-7 interaction. These data suggest that Sld2 association with DNA is required for CMG assembly in S phase.
Highlights
Sld2 is required for the initiation of DNA replication
We identified a mutant of Sld2, Sld2-m1,4, that is defective in Mcm2-7 binding
Sld2 Regulates CMG Helicase Assembly—We identified a mutant of Sld2, Sld2-K416E,R435E,R436E (Sld2-m1,4), that is defective in binding Mcm2-7 in vitro (Fig. 1)
Summary
Sld is required for the initiation of DNA replication. Results: An Sld2-Mcm binding mutant exhibits premature helicase assembly, and an Sld2-DNA binding mutant displays no helicase assembly. Of the Cdc45-Mcm2-7-GINS (CMG) replicative helicase complex must be regulated to ensure that DNA unwinding is coupled with DNA synthesis. We study a mutant of Sld that is defective in binding DNA, sld2-DNA, and find that sld2DNA cells exhibit no GINS-Mcm interaction These data suggest that Sld association with DNA is required for CMG assembly in S phase. RPA-chromatin immunoprecipitation assays of cells exposed to hydroxyurea demonstrate that RPA is not recruited to origins in S phase in sld2-m1,4 mutant cells, unlike wild-type cells These data suggest that Sld2-Mcm interaction is required to restrict the association of GINS with Mcm to S phase. We find that for cells expressing sld2-DNA, GINS does not associate with Mcm2-7 These data suggest that Sld2-ssDNA interaction is essential to allow GINS to bind Mcm during S phase
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