Mutational studies of Pa-AGOG DNA glycosylase from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum.
In all organisms studied to date, 8-oxoguanine (GO), an important oxidation product of guanine, is removed by highly conserved GO DNA glycosylases. The hyperthermophilic crenarchaeon Pyrobaculum aerophilum encodes a GO DNA glycosylase, Pa-AGOG (Archaeal GO DNA glycosylase) which has become the founding member of a new family within the HhH-GPD superfamily of DNA glycosylases based on unique structural and functional characteristics. In this study, we made quantitative measurements of the DNA glycosylase activity of Pa-AGOG wild type and some engineered variants under single turnover conditions. The mutagenesis study includes residues Trp222 (W222A and W222F), Trp69 (W69F), Gln31 (Q31S) and Lys147 (K147Q) all of which are involved in GO recognition and Asp172 (D172N and D172Q) and Lys140 (K140Q) that are involved in catalysis. Pa-AGOG prefers GO/G mispairs for both base excision and base excision/beta-lyase activities. The mutagenesis studies show that base-stacking between GO and Trp222 is very important for recognition. The contact between Trp69 and the 8-oxo group was found to be dispensable, while that to N7 by Gln31 is indispensable for GO recognition. In contrast to human OGG1 the catalytic mutant, D172Q did not show detectable glycosylase activity. Pa-AGOG mutants K140Q, D172N and D172Q did bind GO containing single-stranded DNA more tightly than double-stranded DNA containing a GO/C base pair. Our studies confirm and extend the unique characteristics of Pa-AGOG, which distinguish it from other mesophilic and thermostable GO DNA glycosylases.
Lingaraju
Gondichatnahalli M
GM
Biomolecular Research, Paul Scherrer Institut, CH-5232 Villigen, Switzerland.
Prota
Andrea E
AE
Winkler
Fritz K
FK
D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D002942:Circular Dichroism; D045647:DNA Glycosylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D004795:Enzyme Stability; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D041543:Pyrobaculum / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013696:Temperature
D019843:Archaeal Proteins; D004277:DNA, Single-Stranded; D009841:Oligonucleotides; C024829:8-hydroxyguanine; D006147:Guanine; D045647:DNA Glycosylases