Identification of the Archaeoglobus fulgidus endonuclease III DNA interaction surface using heteronuclear NMR methods.
BACKGROUND
Endonuclease III is the prototype for a family of DNA-repair enzymes that recognize and remove damaged and mismatched bases from DNA via cleavage of the N-glycosidic bond. Crystal structures for endonuclease III, which removes damaged pyrimidines, and MutY, which removes mismatched adenines, show a highly conserved structure. Although there are several models for DNA binding by this family of enzymes, no experimental structures with bound DNA exist for any member of the family.


RESULTS
Nuclear magnetic resonance (NMR) spectroscopy chemical-shift perturbation of backbone nuclei (1H, 15N, 13CO) has been used to map the DNA-binding site on Archaeoglobus fulgidus endonuclease III. The experimentally determined interaction surface includes five structural elements: the helix-hairpin-helix (HhH) motif, the iron-sulfur cluster loop (FCL) motif, the pseudo helix-hairpin-helix motif, the helix B-helix C loop, and helix H. The elements form a continuous surface that spans the active site of the enzyme.


CONCLUSIONS
The enzyme-DNA interaction surface for endonuclease III contains five elements of the protein structure and suggests that DNA damage recognition may require several specific interactions between the enzyme and the DNA substrate. Because the target DNA used in this study contained a generic apurinic/apyrimidinic (AP) site, the binding interactions we observed for A. fulgidus endonuclease III should apply to all members of the endonuclease III family and several interactions could apply to the endonuclease III/AlkA (3-methyladenine DNA glycosylase) superfamily.
Shekhtman
A
A
Department of Physics, University at Albany, SUNY, NY 12222, USA.
McNaughton
L
L
Cunningham
R P
RP
Baxter
S M
SM
D000595:Amino Acid Sequence; D019611:Archaeoglobus fulgidus / Q000201:enzymology*; D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D017931:DNA Primers; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D029968:Escherichia coli Proteins; D004247:DNA; D004706:Endodeoxyribonucleases; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli