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Identification of small molecule inhibitors of the Mcm2-7 replicative helicase, and structural determination of mutants affecting the Mcm2/5 gate

Simon, Nicholas (2014) Identification of small molecule inhibitors of the Mcm2-7 replicative helicase, and structural determination of mutants affecting the Mcm2/5 gate. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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The replication of the eukaryotic genome is a highly regulated process requiring the coordinated efforts of several enzymes to ensure that multiple chromosomes are efficiently replicated once, and only once, per cell cycle. This coordination is largely achieved by loading and activating the eukaryotic replicative helicase, the molecular motor that separates duplex DNA, at different points in the cell cycle. Consistent with this central role in DNA replication’s regulation, the eukaryotic helicase, Mcm2-7, is unique among known helicases in that it is composed of six unique and essential subunits. Prior work has indicated that only a subset of these subunits are required for helicase activity, suggesting a regulatory role for the remainder. It has been posited that two of the subunits, Mcm2 and Mcm5, form an ATP dependent ‘gate’ involved in loading and/or activating the complex. The presence of this gate has been shown biochemically and structurally, but its physiological role remains speculatory. My dissertation research has focused on two areas. The first has been the identification and characterization of quinolone-based inhibitors, with the goal of finding compounds that selectively inhibit the subunits that make up the ‘gate.’ I have found that the fluoroquinolone ciprofloxacin and related compounds are selective for the Mcm2-7 helicase over other helicases and inhibit the complex in both in vitro DNA unwinding assays and in tissue culture. The second area of research has been determining the structure of regulatory mutants we predict have aberrant gate function with transmission electron microscopy and single particle image averaging. I have shown that the regulatory mutant mcm5bob1 biases the complex toward a closed state, potentially allowing for the premature loading of replication factors. I have also demonstrated that a mutation in the Walker B AAA+ ATPase active site motif in the active site between Mcm2 and Mcm6 changes the size of the Mcm2/5 gate, indicating a role for this active site in the regulation of Mcm2-7 topology.


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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 MemberBrodsky, Jeffrey Lbrodsky@pitt.eduBRODSKY
Committee MemberConway, James F.jxc100@pitt.eduJXC100
Committee MemberVandemark, Andrewandyv@pitt.eduANDYV
Committee MemberJen-Jacobson, Lindaljen@pitt.eduLJEN
Date: 30 May 2014
Date Type: Publication
Defense Date: 7 March 2014
Approval Date: 30 May 2014
Submission Date: 17 April 2014
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Number of Pages: 164
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: DNA replication, Mcm2-7, Mcm complex, inhibitors, electron microscopy, fluoroquinolones, helicase, yeast, TEM
Date Deposited: 30 May 2014 12:09
Last Modified: 15 Nov 2016 14:19


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