Hancock, Stephen Paul
(2009)
THE ROLE OF PHOSPHATE NEUTRALIZATION IN ECORV INDUCED DNA BENDING.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
DNA bending by DNA binding proteins is required to facilitate a myriad of essential cellular processes, including genome packaging, the formation of multi-protein complexes necessary for the expression of genetic information, the regulation of gene expression, and the correct positioning of recognition and catalytic elements required for site-specific hydrolysis of DNA. One mechanism proposed for protein induced DNA bending is that protein mediated neutralization of negatively charged phosphates on one face of the DNA produces a collapse of the DNA toward the neutralized surface. In order to further understand the role of phosphate neutralization in DNA bending, I have manipulated the energy required for achieving the 50o axial bend in the EcoRV endonuclease-DNA complex by either removing cationic protein side chains that contact DNA phosphates and/or replacing the charged phosphate with an uncharged methylphosphonate. I present evidence that neutralization of particular phosphates, positioned on the concave face of the bound DNA, can contribute favorably not only to the formation of the EcoRV-recognition complex but also to cleavage of the GATATC site. In addition, synergistic effects are observed when particular combinations of phosphates are neutralized. Fluorescence resonance energy transfer studies show that there is no significant difference in the degree of DNA bending in unmodified and modified complexes, implying that phosphate neutralization modulates the energetic cost of bending rather than the extent of bending. Further, van't Hoff analyses indicate that removal of interphosphate repulsion by phosphate neutralization contributes favorable enthalpy to EcoRV-DNA complex formation, and molecular dynamics simulations show that this favorable enthalpy does not derive from the formation of new contacts between the introduced methyl group and the protein. Taken together, these results support the model that asymmetric phosphate neutralization by a site-specific protein promotes DNA bending. My work marks the first in depth thermodynamic analysis of the impact of phosphate neutralization on protein-induced bending by a DNA bending protein. Further, it shows that this is an energetic strategy employed by proteins to overcome the energetic cost of DNA bending.
Share
| Citation/Export: |
|
| Social Networking: |
|
Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
| Creators | Email | Pitt Username | ORCID  |
|---|
| Hancock, Stephen Paul | sph17@pitt.edu | SPH17 | |
|
| ETD Committee: |
|
| Date: |
28 January 2009 |
| Date Type: |
Completion |
| Defense Date: |
4 August 2008 |
| Approval Date: |
28 January 2009 |
| Submission Date: |
19 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: |
; DNA bending; Methylphosphonates; Phosphate neutralization; Protein-DNA interaction |
| Other ID: |
http://etd.library.pitt.edu/ETD/available/etd-09192008-141347/, etd-09192008-141347 |
| Date Deposited: |
10 Nov 2011 20:02 |
| Last Modified: |
15 Nov 2016 13:50 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/9367 |
Metrics
Monthly Views for the past 3 years
Plum Analytics
Actions (login required)
 |
View Item |
![[feed]](/images/feed-icon-32x32.png){kind=icon}