Structural study of polynucleotidyl transferase family DNA repair enzyme endonuclease V
Abstract
Endonuclease V (EndoV) is a DNA repair enzyme which recognizes a remarkable variety of lesions including deaminated bases, abasic sites and base mismatches. Among these, deaminated inosine is the most physiologically important substrate. EndoV cleaves at the second phosphodiester bond 3' to the lesion, leaving 3' OH and 5' P groups. EndoV contains a characteristic RNase H like core of enzymes from the polynucleotidyl transferase (PNT) superfamily. Besides the RNase H like core, which is a three layer α/β/α structure with the central β sheet sandwiched by a helices on both sides, the PNT family members also share a conserved active site DDE motif. Thermotoga maritima EndoV was crystallized with a 7 base pair DNA duplex containing a damaged inosine nucleotide and a gap at the cleavage site. In order to increase the stability and promote crystallization without base stacking, both ends of this short DNA duplex were capped by a synthetic endcap molecule. The DNA backbone was bent 115° with the abnormal inosine nucleotide flipped out of the DNA helix. Tyr80 protruded into the DNA duplex, and displaced the damaged hypoxanthine base into a nucleotide binding pocket within EndoV. A "Probe-Push-Pull" mechanism was proposed for base-flipping in EndoV. Originally, Asp43, Glu89 and Asp110 were proposed as the catalytic DDE motif of EndoV. However two divalent metal co-factors, coordinating to Asp43, Asp110 and His214, were found in the active site of EndoV/DNA complex structure. Thus, a DDH motif, instead of the canonical DDE motif, was revealed for Thermotoga maritima EndoV. A two-metal catalysis mechanism, with one metal ion activating the water nucleophile and the other stabilizing the pentacovalent transition state and 3' leaving group, was also proposed for EndoV. This EndoV-Damaged DNA structure provides insight into EndoV's role in DNA repair.
Degree
Ph.D.
Advisors
Friedman, Purdue University.
Subject Area
Biophysics
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.