Date of Award
5-2007
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Legacy Department
Biochemistry
Committee Chair/Advisor
Cao, Weiguo
Abstract
The integrity of the genetic information encoded by DNA is essential to all living organisms, yet the reactive bases of DNA are constantly attacked by endogenous and exogenous agents resulting in as many as one million individual molecular lesions per cell per day. Excessive DNA damage or deficiency in DNA repair enzymes may cause cancer, premature aging, and neurodegenerative diseases.
Endonuclease V (Endo V) is a DNA repair enzyme which can recognize all four types of DNA deamination products, specifically, uracil, hypoxanthine, xanthine and oxanine. It was also shown that endo V can recognize mismatches. We screened about 60 mutants of endo V from Thermotoga maritima and found some mutants had altered base preferences for mismatches. Tma endo V Y80A was shown to become a C-specific mismatch endonuclease. G13D mutation in K-ras oncogene which was not recognized by wild type Tma endo V was successfully cleaved by Tma endo V Y80A. This study provides valuable information on base recognition and active site organization of Tma endo V. Tma endo V mutants can be used for cancer mutation scanning and mutation recognition.
In order to further understand the role of Y80 of endo V in base recognition, we substituted the Y80 with sixteen amino acids. Together with three Y80 mutants isolated before, we characterized all nineteen mutants of Tma endo V Y80 using deaminated base-containing DNA substrates and mismatch-containing DNA substrates. This comprehensive amino acid substitution at a single site (Y80) underlines the importance of aromatic ring and hydrogen bond donor capacity in base recognition by endo V, reveals additional Y80 mutants with altered base preferences in mismatch cleavage, and offers new insight on the role of Y80 in base recognition.
Though endo V was shown to be important for repair of deaminated lesions in vivo, its DNA repair pathway remains unknown. In order to understand the DNA repair pathway mediated by endo V, we have developed a cell-free system from Escherichia coli. The preliminary results indicated that the repair patch of endo V mediated DNA repair pathways may consist of a long patch and a short patch repair pathway.
Recommended Citation
Gao, Honghai, "BIOCHEMICAL STUDY OF ENDONUCLEASE V AND ITS APPLICATION IN MUTATION SCANNING" (2007). All Dissertations. 65.
https://open.clemson.edu/all_dissertations/65