Methods to study kinase regulation of the replication fork helicase.
Dbf4-Cdc7 phosphorylation of the Mcm2-7 complex is required for the activation of the replication fork helicase in budding yeast cells. There is a genetic interaction between Dbf4-Cdc7 and Mcm2, and Dbf4-Cdc7 phosphorylates Mcm2 in vitro and in vivo. We initiated a focused study of how Dbf4-Cdc7 phosphorylates Mcm2 in budding yeast, and we also investigated the in vivo implications of this kinase reaction. Described herein are detailed methods for how we conducted biochemical and genetic experiments to dissect the mechanism and function of Dbf4-Cdc7 phosphorylation of Mcm2 in budding yeast cells. The methods are likely applicable to other kinase reactions and studies of replication fork helicases from other organisms.
Kaplan
Daniel L
DL
Vanderbilt University, Department of Biological Sciences, VU Station B, Box 35-1634, Nashville, TN 37235, USA. Daniel.Kaplan@Vanderbilt.Edu
Bruck
Irina
I
D000595:Amino Acid Sequence; D002470:Cell Survival; D004265:DNA Helicases / Q000378:metabolism*; D004261:DNA Replication / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D016415:Sequence Alignment
D029701:Saccharomyces cerevisiae Proteins; D004265:DNA Helicases