Schnable, Brittani
(2024)
Single-molecule studies of thymine DNA glycosylase interacting with DNA.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Base excision repair (BER) is suggested to be the main pathway involved in oxidative DNA demethylation. One such oxidized moiety, 5-formylctyosine (5fC), is recognized and removed by thymine DNA glycosylase (TDG) to generate an abasic site, an intermediate in active demethylation. TDG binds avidly to abasic sites and is product inhibited. Using single molecule fluorescence experiments, we saw TDG slide on unmodified DNA and have specific, stationary binding to 5fC with lifetimes of 7.5 and 72.9 seconds, respectively. Mean squared displacement analysis and a two color TDG experiment indicate that TDG utilizes hopping and sliding in search of a modified base. The catalytically crippled variants, N140A and R275A/L, have a reduced binding lifetime compared to wild type and mean squared displacement (MSD) analysis indicates that R275L/A moves on the DNA with a faster diffusivity. These results indicate that mutating R275, but not N140, interferes with damage recognition by TDG. On DNA containing an undamaged nucleosome, TDG either bypassed, collided by could not bypass or colocalized with the nucleosome after contact. However, truncating the TDG N-terminus significantly reduced the number of interactions with the nucleosome. Additionally, biochemical studies showed that UV-DDB, UV-damaged DNA binding protein the first responder in nucleotide excision repair (NER), can stimulate the turnover of TDG 14-fold as well as displace TDG from an abasic site. Single molecule experiments revealed that TDG and DDB2 colocalize at 5fC. Our findings give insight into how TDG searches for its lesions in long stretches of undamaged DNA and chromatin and indicate that UV-DDB is able to stimulate TDG by facilitated dissociation.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
14 October 2024 |
| Date Type: |
Publication |
| Defense Date: |
17 May 2024 |
| Approval Date: |
14 October 2024 |
| Submission Date: |
31 July 2024 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
141 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Medicine > Molecular Biophysics and Structural Biology |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
TDG, single molecule, Protein-DNA interaction, base excision repair, nucleotide excision repair |
| Date Deposited: |
14 Oct 2024 15:48 |
| Last Modified: |
14 Oct 2024 15:48 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/46790 |
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