Date of Award

12-2008

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Biochemistry

Advisor

Cao, Weiguo

Committee Member

Chen , Chin-Fu

Committee Member

Morris , James C.

Committee Member

Powell , Gary

Abstract

DNA repair enzymes and pathways are diverse and critical for living cells to maintain correct genetic information. Single-strand-selective monofunctional uracil DNA glycosylase (SMUG1) belongs to Family 3 of the uracil DNA glycosylase superfamily. We report that a bacterial SMUG1 ortholog in Geobacter metallireducens (Gme) and the human SMUG1 enzyme are not only uracil DNA glycosylases (UDG), but also xanthine DNA glycosylases (XDG). Mutations at M57 (M57L) and H210 (H210G, H210M, H210N) can cause substantial reductions in XDG and UDG activities. Increased selectivity is achieved in the A214R mutant of Gme SMUG1 and G60Y completely abolishes XDG and UDG activity. Most interestingly, a proline substitution at the G63 position switches the Gme SMUG1 enzyme to an exclusive uracil DNA glycosylase. Mutational analysis and molecular dynamics (MD) simulations of Gme SMUG1 identify important structural determinants in conserved motifs 1 and 2. Our study offers insights on the important role that modulation of conformational flexibility may play in defining specificity and catalytic efficiency.
Endonuclease V is an enzyme that initiates a conserved DNA repair pathway by making an endonucleolytic incision at the 3' side one nucleotide from a deaminated base lesion. This study defines the endonuclease and exonuclease activity in endonuclease V from Thermotoga maritima (Tma) in an assay condition with Mn2+ as a metal cofactor. Tma endonuclease V exhibits inosine-dependent 3'-exonuclease activity. Detailed kinetic analysis using 3'-labeled DNA indicates that Tma endonuclease V also possesses nonspecific 5'-exonuclease activity. The multiplicity of the endonuclease and exonuclease activity is discussed with respect to deaminated base repair.
Biochemical properties of human endonuclease V with respect to repair of deaminated base lesions were reported. We determined repair activities of human endonuclease V on inosine (I)-, xanthosine (X)-, oxanosine (O)- and uridine (U)-containing DNA. Human endonuclease V is most active with inosine-containing DNA; however, with minor activity on xanthosine-containing DNA. Mg2+ and to a much less extent, Mn2+, Ni2+, Co2+ can support the endonuclease activity. Introduction of human endonuclease V into Escherichia coli cells caused two-fold reduction in mutation frequency. This is the first report of deaminated base repair activity from human endonuclease V.

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Biochemistry Commons

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