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 deamina tion 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 u nder physiological conditions, In human cells, the enzymatic activities ini tiating 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 hyd rolytic AP endonuclease (APE/HAP1), respectively. In recent work, we observ ed that BER of the three lesions occurs in human cell extracts with differe nt efficacy, In particular, 8-oxoG is repaired on average 4-fold less effic iently than U, which, in turn, is repaired 7-fold slower than the natural A P 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 immunode tection techniques. Our results show that a single human fibroblast contain s 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 t hree lesions are repaired in vitro. Addition to reaction mixtures of titrat ed amounts of purified hOGG1, UNG and APE/HAP1 variably stimulated the in v itro repair replication of 8-oxoG, U and the AP site respectively and the i ncrease was not always proportional to the amount of added enzyme. We concl ude that the rates of BER depend only in part on cellular levels of initiat ing enzymes.