IDENTIFICATION OF BASE EXCISION REPAIR PATHWAY COMPONENTS IN THE NEMATODE CAENORHABDITIS ELEGANS

Abstract

Carcinogenicity and cytotoxicity are severe consequences to DNA damage. Therefore, several DNA repair pathways function faithfully to preserve the integrity of the genome. Base excision repair (BER) is the major pathway to repair oxidative and alkylating DNA lesions, where enzymes active in the pathway recognize damaged bases and facilitate their replacement. The nematode Caenorhabditis elegans is an attractive model for the study of BER, however, the full pathway has not been fully characterized in C. elegans to this date. In this thesis project, we purified the uracil DNA glycosylase-1 of C. elegans (CeUNG-1), the first enzyme of the BER pathway, as a GST tagged protein and used it as an affinity tag to capture CeUNG-1 interacting partners from N2 wild type C. elegans total protein extract. Mass spectrometry analysis indicates that CeUNG-1 interacts with the ribosomal protein S3 (CeS3), which possesses a widely conserved AP lyase activity, and a unique 8-oxoguanine glycosylase activity in the fruit fly Drosophila melanogaster. Protein-protein docking studies indicate that CeUNG-1 interacts mainly with the middle domain of CeS3, and sequence alignment indicates a 75% shared identity with human S3. CeS3 lacks a critical glutamine residue at position 59 that is essential for 8-oxoGuanine DNA glycosylase activity. Mass spectrometry also denotes an interaction of CeUNG-1 with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which has a confirmed involvement in BER in other eukaryotes. In the second chapter of this work, we sought to develop a simple and convenient in vitro assay for detecting and characterizing DNA nuclease activity in the protein extract of wild type C. elegans using activity gel electrophoresis. However, several optimization steps need to implemented before reproducible results can be seen.

Date of Award	2021
Original language	American English
Awarding Institution	HBKU College of Health & Life Sciences