Functional changes in a novel uracil-DNA glycosylase determined by mutational analyses.
Uracil-DNA glycosylase (UDG) is a ubiquitous enzyme found in bacteria and eukaryotes, which removes uracil residues from DNA strands. Methanococcus jannaschii UDG (MjUDG), a novel monofunctional glycosylase, contains a helix-hairpin-helix (HhH) motif and Gly/Pro rich loop (GPD region), which is important for catalytic activity; it shares these features with other glycosylases such as endonuclease III. First, to examine the role of two conserved amino acid residues (Asp150 and Tyr152) in the HhH-GPD region of MjUDG, mutant MjUDG proteins were constructed, in which Asp 150 was replaced with either Glu or Trp (D150E and D150W), Tyr152 was replaced with either Glu or Asn (Y152E and Y152N). Mutant D150W completely lacked DNA glycosylase activity, whereas D150E displayed reduced activity of about 70% of the wild type value. However, the mutants Y152E and Y152N retained unchanged levels of UDG activity. We also replaced Glu132 in the HhH motif with a lysine residue equivalent to Lys120 in endonuclease III. This mutation converted the enzyme into a bifunctional glycosylase/AP lyase capable of both removing uracil at a glycosylic bond and cleaving the phosphodiester backbone at an AP site. Mutant E132K catalyzes a beta-elimination reaction at the AP site via uracil excision and forms a Schiff base intermediate in the form of a protein-DNA complex.
Im
E K
EK
Yonsei Research Institute of Aging Science, Yonsei University, Seoul 120-749, South Korea.
Han
Y S
YS
Chung
J H
JH
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004260:DNA Repair / Q000235:genetics; D019641:DNA, Archaeal / Q000235:genetics; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017015:Methanococcales / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D016415:Sequence Alignment; D014498:Uracil / Q000378:metabolism; D051981:Uracil-DNA Glycosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D019843:Archaeal Proteins; D019641:DNA, Archaeal; D014498:Uracil; D051981:Uracil-DNA Glycosidase; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase