Mechanism of stimulation of the DNA glycosylase activity of hOGG1 by the major human AP endonuclease: bypass of the AP lyase activity step.
The generation of reactive oxygen species in the cell provokes, among other lesions, the formation of 8-oxo-7,8-dihydroguanine (8-oxoG) in DNA. Due to mispairing with adenine during replication, 8-oxoG is highly mutagenic. To minimise the mutagenic potential of this oxidised purine, human cells have a specific 8-oxoG DNA glycosylase/AP lyase (hOGG1) that initiates the base excision repair (BER) of 8-oxoG. We show here that in vitro this first enzyme of the BER pathway is relatively inefficient because of a high affinity for the product of the reaction it catalyses (half-life of the complex is >2 h), leading to a lack of hOGG1 turnover. However, the glycosylase activity of hOGG1 is stimulated by the major human AP endonuclease, HAP1 (APE1), the enzyme that performs the subsequent step in BER, as well as by a catalytically inactive mutant (HAP1-D210N). In the presence of HAP1, the AP sites generated by the hOGG1 DNA glycosylase can be occupied by the endonuclease, avoiding the re-association of hOGG1. Moreover, the glycosylase has a higher affinity for a non-cleaved AP site than for the cleaved DNA product generated by HAP1. This would shift the equilibrium towards the free glycosylase, making it available to initiate new catalytic cycles. In contrast, HAP1 does not affect the AP lyase activity of hOGG1. This stimulation of only the hOGG1 glycosylase reaction accentuates the uncoupling of its glycosylase and AP lyase activities. These data indicate that, in the presence of HAP1, the BER of 8-oxoG residues can be highly efficient by bypassing the AP lyase activity of hOGG1 and thus excluding a potentially rate limiting step.
Département de Radiobiologie et Radiopathologie, Commissariat à l'Energie Atomique, UMR217 CNRS-CEA, BP6, F92265 Fontenay aux Roses, France.
A E
AE
Vidal
I D
ID
Hickson
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S
Boiteux
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JP
Radicella
D019943:Amino Acid Substitution; D001665:Binding Sites; D019757:Carbon-Oxygen Lyases / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043382:DNA-Formamidopyrimidine Glycosylase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009154:Mutation; D009699:N-Glycosyl Hydrolases / Q000737:chemistry / Q000378:metabolism*; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding
D009841:Oligonucleotides; C024829:8-hydroxyguanine; D006147:Guanine; D043223:Deoxyribonuclease IV (Phage T4-Induced); D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; D019757:Carbon-Oxygen Lyases; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase