Determination of active site residues in Escherichia coli endonuclease VIII.
Endonuclease VIII from Escherichia coli is a DNA glycosylase/lyase that removes oxidatively damaged bases. EndoVIII is a functional homologue of endonuclease III, but a sequence homologue of formamidopyrimidine-DNA glycosylase (Fpg). Using multiple sequence alignments, we have identified six target residues in endoVIII that may be involved in the enzyme's glycosylase and/or lyase functions: the N-terminal proline, and five acidic residues that are completely conserved in the endoVIII-Fpg proteins. To investigate the contribution of these residues, site-directed mutagenesis was used to create seven mutants: P2T, E3D, E3Q, E6Q, D129N, D160N, and E174Q. Each mutant was assayed both for lyase activity on abasic (AP) sites and for glycosylase/lyase activity on 5-hydroxyuracil, thymine glycol, and gamma-irradiated DNA with multiple lesions. The P2T mutant did not have lyase or glycosylase/lyase activity but could efficiently form Schiff base intermediates on AP sites. E6Q, D129N, and D160N behaved essentially as endoVIII in all assays. E3D, E3Q, and E174Q retained significant AP lyase activity but had severely diminished or abolished glycosylase/lyase activities on the DNA lesions tested. These studies provide detailed predictions concerning the active site of endoVIII.
Burgess
Sarah
S
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555-1071, USA.
Jaruga
Pawel
P
Dodson
M L
ML
Dizdaroglu
Miral
M
Lloyd
R Stephen
RS
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D017931:DNA Primers / Q000737:chemistry; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004305:Dose-Response Relationship, Drug; D004706:Endodeoxyribonucleases / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D018698:Glutamic Acid / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013941:Thymine / Q000031:analogs & derivatives* / Q000737:chemistry; D014498:Uracil / Q000031:analogs & derivatives* / Q000737:chemistry
D017931:DNA Primers; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C029389:thymine glycol; D001224:Aspartic Acid; D018698:Glutamic Acid; C045975:5-hydroxyuracil; D014498:Uracil; D011392:Proline; D004706:Endodeoxyribonucleases; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli; D013941:Thymine; D010100:Oxygen