Raja, Sripriya
(2024)
Understanding the role of UV-DDB in the SMUG1-mediated repair of oxidative DNA damage.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
UV-damaged DNA-binding protein (UV-DDB) is a heterodimeric protein, consisting of DDB1 and DDB2 subunits, that works to recognize DNA lesions induced by UV damage during global genome nucleotide excision repair (GG-NER). Our laboratory previously discovered a non-canonical role for UV-DDB in the processing of 8-oxoG, by stimulating 8-oxoG glycosylase, OGG1, activity 3-fold, MUTYH activity 4-5-fold, and APE1 (apurinic/apyrimidinic endonuclease 1) activity 8-fold. 5-hydroxymethyl-deoxyuridine (5-hmdU) is an important oxidation product of thymidine which is removed by single-strand selective monofunctional DNA glycosylase (SMUG1). Biochemical experiments with purified proteins indicated that UV-DDB stimulates the excision activity of SMUG1 on several substrates by 4-5-fold. Electrophoretic mobility shift assays indicated that UV-DDB displaced SMUG1 from abasic site products. Single-molecule analysis revealed that UV-DDB decreases the half-life of SMUG1 on DNA by more than 8-fold. Immunofluorescence experiments demonstrated that cellular treatment with 5-hmdU (5 µM for 15 min), which is incorporated into DNA during replication, produces discrete foci of DDB2-mCherry, which co-localize with SMUG1-GFP. Proximity ligation assays supported a transient interaction between SMUG1 and DDB2 in cells. Poly (ADP)-ribose accumulated after 5-hmdU treatment, which was abrogated with SMUG1 and DDB2 knockdown. Moreover, we observed changes in cell bioenergetics during active 5-hmdU repair. These data support a novel role for UV-DDB in the processing of the oxidized base, 5-hmdU.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
|
| Date: |
16 September 2024 |
| Date Type: |
Publication |
| Defense Date: |
7 September 2023 |
| Approval Date: |
16 September 2024 |
| Submission Date: |
20 October 2023 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
184 |
| 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: |
DNA damage, UV-DDB, SMUG1, oxidative damage, Base excision repair, nucleotide excision repair |
| Date Deposited: |
16 Sep 2024 19:03 |
| Last Modified: |
16 Sep 2024 19:03 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/45451 |
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