Ghodke, Harshad
(2013)
Single Molecule studies of Damage Recognition by the human UV-damaged DNA-binding protein (UV-DDB).
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Nucleotide excision repair (NER) is a DNA repair pathway that processes helix distorting lesions in DNA. In humans, lesions such as UV-induced photoproducts are recognized by the UV-damaged DNA binding protein (UV-DDB). How human DNA repair proteins survey the genome for UV-induced photoproducts remains a poorly understood aspect of the initial damage recognition step in nucleotide excision repair (NER). Specifically, the transport mechanisms employed by UV-DDB, as well as, the stoichiometry of UV-DDB on physiologically relevant DNA substrates containing DNA damage remain unclear.
To understand damage recognition by UV-DDB, we performed single molecule experiments, which revealed that the human UV-damaged DNA binding protein (UV-DDB) samples damage in DNA primarily via a three dimensional search mechanism. We found that UV-DDB displays a remarkable heterogeneity in the kinetics of damage recognition. Our results indicate that UV-DDB examines sites on DNA in discrete steps prior to forming long-lived, non-motile (DDB1-DDB2)2 dimers at sites of damage.
To understand structure-function relationships governing DNA damage recognition by UV-DDB, we tested the xeroderma pigmentosum group E (XP-E) causing K244E mutant of DDB2 found in patient XP82TO. We found that K244E DDB2 supported UV-DDB dimerization but was found to slide on DNA and failed to stably engage lesions. These findings provide molecular insight into the loss of damage discrimination observed in this XP-E patient.
Here we propose a framework for a conformational proofreading mechanism for specific damage recognition by UV-DDB.
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Details
| Item Type: |
University of Pittsburgh ETD
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| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
10 December 2013 |
| Date Type: |
Publication |
| Defense Date: |
22 November 2013 |
| Approval Date: |
10 December 2013 |
| Submission Date: |
9 December 2013 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
198 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Medicine > Pharmacology and Chemical Biology |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
UV-DDB, Nucleotide excision repair, single particle tracking, DNA damage recognition, conformational proofreading, xeroderma pigmentosum |
| Date Deposited: |
10 Dec 2013 18:02 |
| Last Modified: |
19 Dec 2016 14:41 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/20245 |
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