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Identification and characterization of novel proteins involved in initiation of DNA replication in budding yeast

Authors Zhai, Yuanliang
Issue Date 2010
Summary DNA replication in eukaryotic cells is tightly regulated to ensure faithful inheritance of the genetic material. While the replication origins and many replication-initiation proteins in the model organism Saccharomyces cerevisiae have been identified and extensively investigated, the detailed mechanism that controls the initiation of DNA replication is still not well understood. It is likely that some factors involved in or regulating the initiation process have not been discovered. To identify novel DNA replication-initiation proteins and their regulators, I, collaborated with others in our lab, developed a sensitive and comprehensive phenotypic screen by combining several established genetic strategies including plasmid loss assays with plasmids containing a single versus multiple replication origins and colony color sectoring assays. Dozens of mutants in previously known initiation proteins and identified several novel factors, including Ctf1p, Ctf3p, Ctf4p, Ctf18p, Adk1p, Cdc14p and Cdc60p, whose mutants lose a single replication origin-containing plasmid at high rates but lose a multiple replication origins-carrying plasmid at lower rates, were identified. Replication licensing is achieved through sequential loading of several replication-initiation proteins onto replication origins to form pre-replicative complexes (pre-RCs). On the other hand, unscheduled replication licensing is prevented by cyclin-dependent kinases (CDKs) through inhibitory phosphorylations of multiple initiation proteins. It is known that CDK inactivation during mitotic exit promotes pre-RC formation for the next cell cycle. However, the essentiality of removing the inhibitory phosphorylations on the initiation proteins and the acting phosphatase(s) are elusive. In this thesis, I, collaborated with others in our lab, demonstrated in budding yeast that Cdc14p dephosphorylates Orc2p, Orc6p, Cdc6p and Mcm3p to restore their competence for pre-RC assembly. Cells without functional Cdc14p fail to dephosphorylate initiation proteins and to form pre-RCs even when CDK activities are suppressed and cannot replicate DNA in mitotic rereplication systems, while pulsed ectopic expression of Cdc14p in mitotic cells results in efficient pre-RC assembly and DNA rereplication. Furthermore, Cdc14p becomes dispensable for DNA rereplication in mitotic cells with combined non-phosphorylatable / phosphorylation-insensitive alleles of the initiation proteins. These data unravel the essential role of Cdc14p in replication licensing besides its established functions for mitotic exit, providing new insight into the intricate regulation of DNA replication by the interplay between CDKs and the Cdc14p phosphatase. Further characterization of the proteins identified from the IDR screen would surely advance our understanding of the regulation of initiation of DNA replication during the cell cycle.
Note Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2010
Language English
Format Thesis
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