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Title: Molecular mechanism of regulation of the cellular protein levels of endonuclease III homologue (NTH1) in response to DNA damage
Author: Williams, Sarah
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2018
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Deoxyribonucleic acid (DNA) is the store of genetic material, needed for cellular survival and replication. Cellular DNA is under constant attack from genotoxic agents, arising endogenously or exogenously in relation to the cell. Maintaining the stability of the genome is imperative to ensure accurate inheritance of the genetic code for future progeny and ensures that crucial biological processes are undisturbed. To uphold the integrity of the genome, cells have developed numerous DNA surveillance and repair mechanisms. The base excision repair (BER) pathway is one of the pathways that has evolved to remove minor types of DNA damage. The main sub-pathway of BER involves recognition and removal of the oxidised DNA base lesion, incision of the phosphodiester backbone, followed by insertion of the correct complementary nucleotide, before the nick in the DNA backbone is restored. During BER, the recognition of damaged bases relies on DNA glycosylase enzymes. Human endonuclease III (NTH1) is a DNA glycosylase enzyme which specifically recognises oxidised base lesions, caused by reactive oxygen species (ROS). Crucially, NTH1 excises thymine glycol (Tg), which is a particularly mutagenic base lesion. Regulation of enzymes implicated in the BER pathway is important to prevent excessive DNA damage. Reports have demonstrated the importance of post translational modifications (PTMs) in controlling the levels, activity and interactivity of BER proteins. Ubiquitylation is a PTM that has been implicated in the regulation of several BER enzymes. Ubiquitylation is completed by the ubiquitin proteasome pathway (UPP); whereby E3 ligase enzymes attach of moieties of ubiquitin to lysine residues of substrate proteins. The attachment of multiple moieties of ubiquitin (polyubiquitylation) is associated with the regulation of protein levels, whereas, the attachment of singular subunits of ubiquitin (monoubiquitylation) can have variable consequences. Despite this understanding, evidence of PTMs that target human NTH1 are deficient. A proteomic study demonstrated that NTH1 is subject to ubiquitylation dependent regulation, but the specific UPP enzymes involved were not identified. The overarching aim of this project was to understand the molecular mechanisms employed by human cells to regulate levels of NTH1 via ubiquitylation, with specific emphasis on discovering the E3 ligase enzymes involved. Using a well refined purification technique, human cell extracts were fractionated using a series of column chromatography columns. Candidate E3 ligase activity was examined using in vitro ubiquitylation assays of chromatography fractions, with recombinant NTH1 as the substrate protein. Eventually, mass spectrometry analysis of an isolated ubiquitylated fraction, identified tripartite motif containing 26 (TRIM26) as the only candidate E3 ligase enzyme present in the active fraction. We strengthened this finding by repeating ubiquitylation assays using recombinant TRIM26. Together, these findings identify TRIM26 as the major E3 in human cell extracts that catalyses the ubiquitylation of NTH1. Following this, site directed mutagenesis identified lysine 67 as the major site of TRIM26 dependent ubiquitylation of NTH1. Aside to this, the cellular implications of TRIM26 dependent regulation of NTH1 were investigated. Elevated levels of cellular NTH1 following proteasome inhibition confirmed that NTH1 levels may be regulated in cells by ubiquitylation dependent degradation. However, depletion of cellular TRIM26 via siRNA had no significant impact on the steady state levels of the glycosylase. Fractionation of cellular extracts confirmed that cellular NTH1 is located primarily in the nucleus and may be associated with chromatin. Depletion of TRIM26 resulted in no alteration in the cellular distribution of the glycosylase. Since TRIM26 dependent ubiquitylation did not appear to regulate the steady state levels of NTH1, further examination using hydrogen peroxide as a DNA damaging agent showed that DNA damage responsive levels of NTH1 protein expression increased following TRIM26 depletion. The clonogenic assay demonstrated that cells had increased survival capacity in the absence of TRIM26. Importantly, this observation was recapitulated with a partial overexpression of NTH1. Similarly, the alkaline single gel electrophoresis (comet) assay, in combination with an siRNA mediated depletion of TRIM26, concluded that cells with reduced TRIM26 levels have improved ability to manage oxidative stress. Once more, a similar level of improved DNA repair kinetics could be achieved by partially overexpressing NTH1. Overall, I successfully purified and identified TRIM26 as the major E3 ligase that ubiquitylates NTH1. The major site of TRIM26 dependent ubiquitylation is lysine 67; as substitution of this residue substantially impeded in vitro ubiquitylation via TRIM26. Cellular studies confirmed that levels of NTH1 may be regulated by ubiquitylation dependent degradation, although, TRIM26 dependent ubiquitylation was not implicated in the regulation of steady state levels of NTH1. Rather, it appears that TRIM26 may be implicated in the regulation of DNA damage responsive levels of NTH1. Interestingly, a separate investigation in our laboratory previously identified TRIM26 as one of the major E3 ligase enzymes involved in regulation of the steady state levels of another BER glycosylase, endonuclease VIII-like protein 1 (NEIL1). Excitingly, the outcomes of this work have now been peer-reviewed and accepted for publication in the Molecular and Cellular Biology scientific journal (see appendix). In summary, an increasingly dynamic role of TRIM26 is now becoming apparent; whereby, the E3 ligase is involved in the regulation of multiple BER glycosylases with different effects in relation to the DNA damage response. Despite this increased perception, the cellular mechanism which dictates the outcome of ubiquitylation of either glycosylase under the regulation of TRIM26 remains to be fully understood and demands further inspection.
Supervisor: Parsons, Jason Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral