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Title page for ETD etd-07082015-230321


Type of Document Dissertation
Author Dhingra, Nalini
Author's Email Address nalini.dhingra@vanderbilt.edu
URN etd-07082015-230321
Title Elucidating the role of Dpb11 in replication initiation
Degree PhD
Department Biological Sciences
Advisory Committee
Advisor Name Title
Dr. Todd Graham Committee Chair
Keywords
  • initiation
  • replication fork
  • kinase
  • helicase
  • replication
Date of Defense 2015-05-06
Availability unrestricted
Abstract
Initiation of DNA Replication is a highly regulated process and is characterized by the conversion of inactive Mcm2-7 double hexamer around dsDNA to an active helicase consisting of Mcm2-7 single hexamer which encircles ssDNA along with Cdc45 and GINS. This transition of inactive Mcm2-7 around dsDNA to the active helicase around ssDNA involves several unknown mechanisms. Many proteins like, Sld2, Sld3, Dpb11, Mcm10, have been reported to be essential for replication initiation. Dpb11 is one of these essential proteins that functions in the initiation of DNA replication. Dpb11 binds to S-CDK phosphorylated Sld2 and Sld3 to form a ternary complex during S phase. These interactions are essential for the CDK-dependent activation of DNA replication in budding yeast. Studies have also indicated that chromosomal replication initiates by a fundamentally similar process in all eukaryotes, with Dpb11 being an evolutionary conserved protein. Our studies using purified proteins from budding yeast show that Dpb11 alone binds to Mcm2-7, and Dpb11 also competes with GINS for binding to Mcm2-7. Furthermore, Dpb11 binds directly to single-stranded DNA (ssDNA), and ssDNA inhibits Dpb11 interaction with Mcm2-7. We also found that Dpb11 can recruit Cdc45 to Mcm2-7. We identified a mutant of the BRCT4 motif of Dpb11 that remains bound to Mcm2-7 in the presence of ssDNA (dpb11-m1,m2,m3,m5), and this mutant exhibits a DNA replication defect when expressed in budding yeast cells. Expression of this mutant results in increased interaction between Dpb11 and Mcm2-7 during S phase, impaired GINS interaction with Mcm2-7 during S phase, and decreased RPA interaction with origin DNA during S phase. We propose a model wherein Dpb11 first recruits Cdc45 to Mcm2-7. Dpb11, while bound to Cdc45-Mcm2-7, can block the interaction between GINS and Mcm2-7. Upon the extrusion of ssDNA from the central channel of Mcm2-7, Dpb11 dissociates from Mcm2-7 and Dpb11 binds to ssDNA, thereby allowing GINS to bind to Cdc45-Mcm2-7. Finally, we propose that Dpb11 functions with Sld2 and Sld3 to help control the assembly of the replication fork helicase.
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