Stimulation of human 8-oxoguanine-DNA glycosylase by AP-endonuclease: potential coordination of the initial steps in base excision repair.
8-Oxoguanine-DNA glycosylase 1 (OGG1), with intrinsic AP lyase activity, is the major enzyme for repairing 7,8-dihydro-8-oxoguanine (8-oxoG), a critical mutagenic DNA lesion induced by reactive oxygen species. Human OGG1 excised the damaged base from an 8-oxoG. C-containing duplex oligo with a very low apparent k(cat) of 0.1 min(-1) at 37 degrees C and cleaved abasic (AP) sites at half the rate, thus leaving abasic sites as the major product. Excision of 8-oxoG by OGG1 alone did not follow Michaelis-Menten kinetics. However, in the presence of a comparable amount of human AP endonuclease (APE1) the specific activity of OGG1 was increased approximately 5-fold and Michaelis-Menten kinetics were observed. Inactive APE1, at a higher molar ratio, and a bacterial APE (Nfo) similarly enhanced OGG1 activity. The affinity of OGG1 for its product AP.C pair (K:(d) approximately 2.8 nM) was substantially higher than for its substrate 8-oxoG.C pair (K:(d) approximately 23. 4 nM) and the affinity for its final ss-elimination product was much lower (K:(d) approximately 233 nM). These data, as well as single burst kinetics studies, indicate that the enzyme remains tightly bound to its AP product following base excision and that APE1 prevents its reassociation with its product, thus enhancing OGG1 turnover. These results suggest coordinated functions of OGG1 and APE1, and possibly other enzymes, in the DNA base excision repair pathway.
Hill
J W
JW
Sealy Center for Molecular Science, Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX 77555, USA.
Hazra
T K
TK
Izumi
T
T
Mitra
S
S
D000626:Aminopeptidases / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D001894:Borohydrides / Q000037:antagonists & inhibitors / Q000494:pharmacology; D019757:Carbon-Oxygen Lyases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D003596:Cytosine / Q000378:metabolism; D018736:DNA Adducts / Q000378:metabolism; D004260:DNA Repair / Q000187:drug effects; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043382:DNA-Formamidopyrimidine Glycosylase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009154:Mutation / Q000235:genetics; D009699:N-Glycosyl Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity / Q000187:drug effects
D001426:Bacterial Proteins; D001894:Borohydrides; D018736:DNA Adducts; D029968:Escherichia coli Proteins; D029701:Saccharomyces cerevisiae Proteins; C024829:8-hydroxyguanine; D006147:Guanine; C025364:sodium borohydride; D003596:Cytosine; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli; D000626:Aminopeptidases; C019689:APE1 protein, S cerevisiae; D019757:Carbon-Oxygen Lyases; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase