Rates of base excision repair are not solely dependent on levels of initiating enzymes.
The oxidized base 8-oxo-7,8-dihydroguanine (8-oxoG), the product of deamination of cytosine uracil (U), and the sites of base loss [abasic (AP) sites] are among the most frequent mutagenic lesions formed in the human genome under physiological conditions. In human cells, the enzymatic activities initiating DNA base excision repair (BER) of 8-oxoG, U and AP sites are the 8-oxoG DNA glycosylase (hOGG1), the U-DNA glycosylase (UNG) and the major hydrolytic AP endonuclease (APE/HAP1), respectively. In recent work, we observed that BER of the three lesions occurs in human cell extracts with different efficacy. In particular, 8-oxoG is repaired on average 4-fold less efficiently than U, which, in turn, is repaired 7-fold slower than the natural AP site. To discriminate whether the different rates of repair may be linked to different expression of the initiating enzymes, we have determined the amount of hOGG1, UNG and APE/HAP1 in normal human cell extracts by immunodetection techniques. Our results show that a single human fibroblast contains 123 000 +/- 22 000 hOGG1 molecules, 178 000 +/- 20 000 UNG molecules and 297 000 +/- 50 000 APE/HAP1 molecules. These limited differences in enzyme expression levels cannot readily explain the different rates at which the three lesions are repaired in vitro. Addition to reaction mixtures of titrated amounts of purified hOGG1, UNG and APE/HAP1 variably stimulated the in vitro repair replication of 8-oxoG, U and the AP site respectively and the increase was not always proportional to the amount of added enzyme. We conclude that the rates of BER depend only in part on cellular levels of initiating enzymes.
DNA Repair Unit, Mutagenesis Laboratory, Istituto Nazionale Ricerca Cancro, Largo Rosanna Benzi n. 10, 16132 Genova, Italy.
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E
Cappelli
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Hazra
J W
JW
Hill
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Slupphaug
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Bogliolo
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Frosina
D020137:Base Pair Mismatch; D019757:Carbon-Oxygen Lyases / Q000378:metabolism*; D045647:DNA Glycosylases; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D006801:Humans; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D011994:Recombinant Proteins; D043223:Deoxyribonuclease IV (Phage T4-Induced); D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D019757:Carbon-Oxygen Lyases; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase