Hughes, Jonathan
(2019)
Ubiquitylation-dependent regulation of 8-oxoguanine DNA glycosylase (OGG1).
PhD thesis, University of Liverpool.
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Abstract
The base excision repair (BER) pathway is the main DNA repair pathways for the removal and correction of minor DNA base lesions arising from endogenous and exogenous DNA damaging sources. Maintaining the integrity and stability of DNA is imperative for cell survival, correct biological functions and the accurate inheritance of the genome by future progeny. The key to the BER pathway is the tight control and regulation of the enzymes involved in order to avoid excessive DNA damage linked with mutagenesis and the development of human diseases such as cancer. It has been widely reported and demonstrated that management of protein levels, interactions and enzymatic activity through posttranslational modifications (PTMs) is highly important for BER regulation. Ubiquitylation, which involves the addition of small protein moieties called ubiquitin, is a PTM that has been shown to control multiple members of the BER pathway. During the ubiquitin proteasome pathway, specific E3 ubiquitin ligase enzymes attach the ubiquitin molecules to the lysine residues of target protein substrates. Recent studies have shown that ubiquitylation catalysed by specific E3 ubiquitin ligases is implicated in the regulation of the DNA glycosylases that excise DNA base lesions, such as endonuclease VIII-like protein 1 (NEIL1) and endonuclease III-like protein 1 (NTH1). The aim of the study described in this thesis was to identify the E3 ligase(s) that targets 8-oxoguanine DNA glycosylase (OGG1) and discover how OGG1 is regulated through ubiquitylation. Here described within, neural precursor cell expressed, developmentally down-regulated 4-like (NEDD4L) was identified as the major E3 ubiquitin ligase enzyme targeting OGG1 for ubiquitylation. This was achieved through the fractionation of human whole cell extracts by sequential column chromatography columns in combination with an in vitro ubiquitylation assay using recombinant His-tagged OGG1 as the substrate. Purified recombinant His-tagged NEDD4L was shown to be capable of ubiquitylating OGG1 in vitro and the site of ubiquitylation was mapped to lysine 341 using a site-directed mutagenesis approach. In cultured U2OS cells, siRNA-mediated depletion of NEDD4L protein levels had no significant impact on the cellular steady state stability of OGG1 protein levels in unstressed cells but demonstrated that the E3 ligase modulates OGG1 protein in response to DNA damage induced by ionising radiation. Clonogenic assays further demonstrated that increased stabilisation of OGG1 following NEDD4L siRNA treatment caused elevated cell sensitivity post-irradiation, which can be phenocopied by transient overexpression of OGG1. Taken together, these results highlight the importance of NEDD4L in controlling the careful balance of OGG1 protein levels and activity required for cell survival in the cellular response to oxidative DNA damage and provide new mechanistic insights into the regulation of the BER pathway.
| Item Type: | Thesis (PhD) |
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| Divisions: | Faculty of Health and Life Sciences > Institute of Life Courses and Medical Sciences > School of Medicine |
| Depositing User: | Symplectic Admin |
| Date Deposited: | 04 Feb 2020 16:14 |
| Last Modified: | 19 Jan 2023 00:12 |
| DOI: | 10.17638/03066931 |
| Supervisors: |
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| URI: | https://livrepository.liverpool.ac.uk/id/eprint/3066931 |
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