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Download or read book Localization of Cdc7 Protein Kinase During DNA Replication in Saccharomyces Cerevisiae written by Daniel C. Rossbach and published by . This book was released on 2017 with total page 171 pages. Available in PDF, EPUB and Kindle. Book excerpt: Dbf4-Dependent Kinase (DDK) is a conserved serine-threonine protein kinase essential for initiation of DNA replication during S phase of the cell cycle. DDK is composed of a regulatory subunit, Dbf4, and a catalytic subunit, Cdc7, and is required throughout S phase to initiate replication at origins by phosphorylating residues within the Mcm2-7 helicase. While the biological significance of DDK, including kinase activity, substrates, and protein expression has been well characterized, the molecular mechanism through which the kinase associates with chromatin is still unclear. Cdc7 is expressed and bound to chromatin throughout the cell cycle, but there has been little empirical evidence to determine where Cdc7 binds within the genome and more specifically whether it binds directly to origins of replication. The Dbf4 regulatory subunit of DDK has been shown to directly interact with origins of replication, according to one-hybrid screens and chromatin immunoprecipitation with G1-arrested cells. Using a variety of biochemical and genetic techniques, this study begins to identify where Cdc7 is bound within the Saccharomyces cerevisiae genome. Due to its role in phosphorylating the MCM helicase at origins of replication, we hypothesized that the Cdc7 catalytic subunit of DDK is bound to chromatin primarily at origins of replication in a manner that depends on association with the Dbf4 regulatory subunit. The Calling Cards method demonstrates that Cdc7 kinase does not solely bind origins of replication, but rather binds to chromatin across the genome. Furthermore, a point mutation in Cdc7 that renders the kinase inactive was found to have a higher binding affinity than wild-type. A 55 amino acid C-terminal truncation no longer binds Dbf4 and reduces the amount of Cdc7 bound to chromatin, suggesting that Dbf4 potentiates the ability of Cdc7 to bind chromatin. Additionally, Cdc7 is bound near functional and non-functional ACS sites. Chromatin immunoprecipitation experiments show that association of Cdc7 kinase with origins of replication changes throughout the cell cycle. We conclude our results suggest the role of Cdc7 to phosphorylate the MCM helicase does not depend on localization directly at origins of replication, but rather the quantity of Cdc7 bound to chromatin as a result of binding to the Dbf4 regulatory subunit.