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Vijayraghavan, Sriram (2014) REGULATION OF GENOME STABILITY VIA MCM2-7 ATPASE ACTIVE SITES IN SACCHAROMYCES CEREVISIAE. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Genome stability is vital to the survival and health of eukaryotic organisms. Consequently, many complex mechanisms coordinate with each other in an intricate fashion to ensure that genomes are preserved during duplication and its subsequent propagation. Despite the vast number of factors involved in these processes, their coordinated regulation hinges on a few key components. One such factor is the eukaryotic replicative helicase Mcm2-7, which is a multi-subunit enzyme complex that unwinds DNA during S-phase and paves the way for nascent DNA synthesis by the polymerases. As an essential and highly versatile replisome component, Mcm2-7 is well-suited as the ideal hub for the regulation of not only DNA replication but other fork-related activities such as S-phase checkpoints and sister chromatid cohesion. While all members of the Mcm2-7 complex are highly conserved and essential in all eukaryotes, their contributions towards DNA unwinding are unequal and distinct, and the in vivo functions of most of the Mcm ATPase active sites has remained largely unknown. We conducted an in vivo analysis of a viable mcm2 ATPase active site allele in Saccharomyces cerevisiae and found that under conditions of genotoxic stress it is deficient in the DNA replication checkpoint (DRC) activation, upstream of the Rad53/CHK2 effector kinase. Furthermore, this allele also exhibited a peculiar cell-cycle specific DNA damage phenotype and defective sister chromatid cohesion (SCC) under conditions that are normally conducive to growth. Importantly, these phenotypes manifest from an apparent defect in ATP hydrolysis rather than a qualitative reduction in Mcm2 protein abundance, stability or complex integrity. Therefore, our study demonstrates for the first time that Mcm2-7 can coordinate DNA replication with genome stability through discrete ATPase active sites. Curiously, these functions appear to be separable from general replication defects as shown through a different subset of mcm mutants, indicating that different active sites of Mcm2-7 pleiotropically coordinate various aspects of genome integrity during S-phase.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairSchwacha, Anthonyschwacha@pitt.eduSCHWACHA
Committee MemberArndt, Karenarndt@pitt.eduARNDT
Committee MemberBrodsky, Jeffrey Ljbrodsky@pitt.eduJBRODSKY
Committee MemberVandemark, Andrewandyv@pitt.eduANDYV
Committee MemberWoolford,
Date: 25 September 2014
Date Type: Publication
Defense Date: 17 July 2014
Approval Date: 25 September 2014
Submission Date: 13 August 2014
Access Restriction: 1 year -- Restrict access to University of Pittsburgh for a period of 1 year.
Number of Pages: 205
Institution: University of Pittsburgh
Schools and Programs: Dietrich School of Arts and Sciences > Biological Sciences
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: Genome stability, DNA replication, Genetic analysis, molecular motors, helicase, sister chromatid cohesion, checkpoints, DNA damage
Date Deposited: 25 Sep 2014 17:06
Last Modified: 15 Nov 2016 14:23


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