Bochman, Matthew L.
(2009)
The Mcm2-7 complex is the eukaryotic replicative helicase.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Replicative helicases are essential enzymes in DNA replication that separate duplex DNA into single strands to be used as templates by polymerases. The identity of these helicases in prokaryotes and viruses has been known for some time, but there is controversy in defining the eukaryotic replicative helicase. Fifteen years of in vivo work and comparison to other helicases suggests that the minichromosome maintenance proteins two through seven (Mcm2-7 complex) play this role, but in vitro DNA unwinding has never been demonstrated. In order to address this dilemma, I analyzed the biochemical activities of the Saccharomyces cerevisiae Mcm2-7 complex and the contributions of its conserved ATPase motifs to these activities. My findings indicate that Mcm2-7: 1) binds single-stranded DNA with high affinity in an ATP-dependent manner, 2) has six distinct active sites that differentially contribute to its biochemistry, and 3) is sensitive to anionic conditions in vitro. Elucidation of these characteristics led to the discovery that Mcm2-7 helicase activity is stimulated by large anions, and I was able to demonstrate bona fide in vitro DNA unwinding for the first time. The culmination of this work is the view that the Mcm2-7 complex is the true eukaryotic replicative helicase and the emerging view that Mcm2-7 ATPase active sites have evolved specialized functions within the complex.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
| Creators | Email | Pitt Username | ORCID  |
|---|
| Bochman, Matthew L. | mlb36@pitt.edu | MLB36 | |
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| ETD Committee: |
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| Date: |
26 January 2009 |
| Date Type: |
Completion |
| Defense Date: |
4 September 2008 |
| Approval Date: |
26 January 2009 |
| Submission Date: |
8 September 2008 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| 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: |
ATPase; DNA replication; helicase; Mcm2-7 |
| Other ID: |
http://etd.library.pitt.edu/ETD/available/etd-09082008-155516/, etd-09082008-155516 |
| Date Deposited: |
10 Nov 2011 20:01 |
| Last Modified: |
15 Nov 2016 13:50 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/9342 |
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