Scott Baird (Committee Member), Steven Berberich (Committee Member), Ashot Kozak (Committee Member), Michael Leffak (Advisor), Yong-jie Xu (Committee Member)
Doctor of Philosophy (PhD)
The initiation of DNA replication is a highly regulated and coordinated process. To ensure that the entire genome is replicated only once per cell cycle, many replication proteins are assembled on the chromatin in a step-wise and cell cycle dependent manner. This process is controlled by interaction of replication proteins, post-translational modifications of the replication factors, control of cellular localization of the proteins, or replication factor degradation after their function terminates. Two kinases, CDK (cyclin dependent kinase) and DDK (Dbf4/Drf1 dependent kinase), play important roles during the initiation stage of DNA replication. The c-myc DNA unwinding element-binding protein (DUE-B) is an essential replication protein that interacts with both the replicative helicase (minichromosome maintenance (MCM)) and the helicase activator (Cdc45) at mammalian replication origins, and is required to load Cdc45 onto chromatin in Xenopus egg extracts. Here, using co-immunoprecipitation experiments, I show that DUE-B interacts with the protein Treslin/TICRR (TopBP1- interacting, replication stimulating protein/TopBP1-interacting, checkpoint and replication regulator, simply refered to as Treslin) which has dual roles in replication and checkpoint in vivo. Treslin, an orthologue of yeast Sld3, is an essential CDK substrate during replication initiation and DNA damage signaling. Treslin collaborates with TopBP1 in the Cdk2-mediated loading of Cdc45 onto replication origins. The interaction between DUE-B and Treslin does not require the presence of DNA but requires TopBP1, another protein with dual functions in replication and checkpoint. TopBP1, a human homolog of budding yeast Dpb11, is a multi BRCT domain containing protein. It is a scaffolding protein that allows interaction between replication and checkpoint proteins. I show that the interaction between DUE-B and Treslin is cell cycle and checkpoint regulated. Interaction between DUE-B and Treslin increases during mid/late G1 phase when the cells prepare for the initiation of DNA replication, and this interaction is significantly reduced in the presence of DNA damage. The interaction of DUE-B with Treslin is mediated through the C-terminal region of DUE-B, which contains multiple serine and threonine residues. Mutation of all serine and threonine residues to phosphomimic or phosphodeficient residues regulate DUE-B binding to the MCM helicase, but did not affect the interaction between DUE-B and Treslin. I also show that knockdown of Treslin in HeLa cells inhibits chromatin binding of DUE-B, and in reverse experiments knockdown of DUE-B inhibits chromatin binding of Treslin. TopBP1 knockdown did not affect the chromatin binding of DUE-B and Treslin but affected Cdc45 chromatin binding. Both DUE-B and Treslin knockdown reduces the levels of Cdc45 on the chromatin. In a time-course experiment, DUE-B, Treslin and TopBP1 follow similar temporal chromatin binding profiles during mid/late G1 phase and Cdc45 chromatin binding follows the binding of DUE-B, Treslin and TopBP1 to the chromatin. Replicative stress caused by aphidicolin and hydroxyurea also reduced the chromatin binding of DUE-B, Treslin and Cdc45 but not TopBP1. The data presented here show that interaction between DUE-B and Treslin is regulated by signals from the cell cycle to control the initiation of DNA replication when DNA replication occurs normally and in the presence of replicative stress, and that the interaction of DUE-B and Treslin is an important step for the loading of the helicase activator, Cdc45, to activate the replicative helicase.
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Copyright 2016, all rights reserved. My ETD will be available under the "Fair Use" terms of copyright law.