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DNA damage of endogenous origin may significantly contribute to human cancer. A major pathway involved in DNA repair of endogenous damage is DNA base excision repair (BER). BER is rather efficient in human cells but a certain amount of endogenous damage inevitably escapes mending and likely contributes to human carcinogenesis. Apart from some glycosylases that are particularly sluggish (e.g. 8-oxoG DNA glycosylase), recent work suggests that the general rate-limiting steps of BER may be trimming of 2-deoxyribose 5-phosphate in case the process is started by a monofunctional glycosylase or trimming of a 3′-blocking fragment, in case BER is started by a bifunctional glycosylase or in the case of single-strand breaks produced by free radical attack. Overexpression of the 5′-deoxyribophosphodiesterase (dRPase) domain of DNA polymerase β, on the one hand, and of yeast APN1 protein, containing an efficient 3′ repair activity, on the other, may lead to improved BER in mammals. The recently characterized S3 protein of Drosophila, containing both dRPase and 3′-trimming activities, could also be considered for overexpression studies. The possible protecting role of enhanced BER could be investigated in cultured rodent embryonic fibroblasts undergoing spontaneous transformation, a most interesting system that merits rediscovery.

DNA damage of endogenous origin may significantly contribute to human cancer. A major pathway involved in DNA repair of endogenous damage is DNA base excision repair (BER). BER is rather efficient in human cells but a certain amount of endogenous damage inevitably escapes mending and likely contributes to human carcinogenesis. Apart from some glycosylases that are particularly sluggish (e.g. 8-oxoG DNA glycosylase), recent work suggests that the general rate-limiting steps of BER may be trimming of 2-deoxyribose 5-phosphate in case the process is started by a monofunctional glycosylase or trimming of a 3'-blocking fragment, in case BER is started by a bifunctional glycosylase or in the case of single-strand breaks produced by free radical attack. Overexpression of the 5'-deoxyribophosphodiesterase (dRPase) domain of DNA polymerase beta, on the one hand, and of yeast APN1 protein, containing an efficient 3' repair activity, on the other, may lead to improved BER in mammals. The recently characterized S3 protein of Drosophila, containing both dRPase and 3'-trimming activities, could also be considered for overexpression studies. The possible protecting role of enhanced BER could be investigated in cultured rodent embryonic fibroblasts undergoing spontaneous transformation, a most interesting system that merits rediscovery.

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.

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.

The repair of the endogenous lesions 8-oxo-7,8-dihydroguanine (8-oxoG), uracil (U) and natural abasic site (AP site) was investigated using an in vitro base excision repair assay in which a plasmid substrate containing a single lesion at a defined position was repaired by mammalian cell extracts. Repair replication of an 8-oxoG/cytosine base pair performed by normal human cell extracts was ~5-fold less efficient than repair of a U/adenine base pair and, in turn, the latter was repaired ~10-fold less efficiently than an AP site placed in front of an adenine. A similar pattern of repair capacity for the three lesions was observed in Chinese hamster extracts. Repair of 8-oxoG was performed by the one nucleotide insertion pathway only. The lower repair replication ability of 8-oxoG with respect to U was linked to a lower DNA glycosylase (base removal) activity rather than to inability to process the β-elimination cleaved strand left by the AP lyase activity associated with human oxoguanine DNA glycosylase 1. The data show that DNA repair of 8-oxoG is poor in human cells in comparison with other frequent endogenous lesions.

The repair of the endogenous lesions 8-oxo-7,8-dihydroguanine (8-oxoG), uracil (U) and natural abasic site (AP site) was investigated using an in vitro base excision repair assay in which a plasmid substrate containing a single lesion at a defined position was repaired by mammalian cell extracts. Repair replication of an 8-oxoG/cytosine base pair performed by normal human cell extracts was approximately 5-fold less efficient than repair of a U/adenine base pair and, in turn, the latter was repaired approximately 10-fold less efficiently than an AP site placed in front of an adenine. A similar pattern of repair capacity for the three lesions was observed in Chinese hamster extracts. Repair of 8-oxoG was performed by the one nucleotide insertion pathway only. The lower repair replication ability of 8-oxoG with respect to U was linked to a lower DNA glycosylase (base removal) activity rather than to inability to process the beta-elimination cleaved strand left by the AP lyase activity associated with human oxoguanine DNA glycosylase 1. The data show that DNA repair of 8-oxoG is poor in human cells in comparison with other frequent endogenous lesions.