Evidence for the involvement of nucleotide excision repair in the removal of abasic sites in yeast.

Abasic (AP) sites arise in DNA at a substantial rate by spontaneous hydrolysis of the N-glycosylic bond, and it has been estimated that as many as 104 purines are lost spontaneously in a human cell per day (27). AP sites are also formed in DNA as intermediates in base excision repair (BER), which removes damaged bases formed by oxidation and alkylation. The first step of BER involves the action of a DNA glycosylase which catalyzes the hydrolysis of the N-glycosylic bond linking the damaged base to the deoxyribose phosphate backbone. The ensuing AP site is recognized by a class II AP endonuclease which cleaves the phosphodiester backbone on the 5′ side of the AP site, leaving a 3′-hydroxyl group and a 5′-baseless deoxyribose 5′-phosphate residue. Removal of the deoxyribose 5′-phosphate residue, followed by DNA repair synthesis and ligation, completes the repair process (6, 39, 45). Two class II AP endonucleases, Apn1 and Apn2, have been identified in the yeast Saccharomyces cerevisiae. Apn1 represents the major AP endonuclease activity in yeast, and it shares extensive homology with Escherichia coli endonuclease IV (31, 32). Apn2 is an homolog of E. coli exonuclease III and of human HAP1 (REF1) AP endonuclease (3, 23). Genetic and biochemical studies have indicated that Apn1 and Apn2 constitute alternate pathways for the removal of AP sites in yeast (23). In contrast to BER, which removes damaged bases which do not perturb the helical structure of DNA, nucleotide excision repair (NER) removes DNA damages that cause significant distortion of the helix (36). For example, NER removes cyclobutane pyrimidine dimers and (6-4) photoproducts formed by ultraviolet light and is also involved in the removal of intrastrand and interstrand cross-links and bulky adducts formed in DNA upon treatment with a variety of chemical agents. NER is a highly conserved process among eukaryotes from yeast to humans (36). In S. cerevisiae, NER is accomplished via the concerted action of the following: Rad14, RPA, the Rad4-Rad23 complex, and the Rad7-Rad16 complex, all of which function in DNA damage recognition (5, 11, 12, 14–16, 22); TFIIH, which contains the Rad3 and Rad25 DNA helicases, essential for DNA unwinding (41); and the Rad1-Rad10 and Rad2 nucleases (18, 42, 43) which incise the damaged strand on the 5′ and 3′ sides of the lesion, respectively (2, 17). Both in yeast and humans, dual incision by the NER ensemble results in the release of an oligonucleotide fragment ∼25 to 30 nucleotides long (10, 28, 29). In humans, a defect in NER results in xeroderma pigmentosum (XP). XP individuals are extremely sensitive to sunlight, and the frequencies of basal cell carcinoma, squamous cell carcinoma, and melanoma of the skin are increased 1,000-fold or more in these patients (24, 25). XP patients also manifest an increase in the incidence of cancers in sites not exposed to UV radiation, and there is a disproportionate increase in the frequency of malignant neoplasms of the brain and other parts of the central nervous system and extraglossal oral cavity in these individuals (24, 25). These observations have suggested the involvement of NER in the removal of non-UV-related DNA damage. Because AP sites are formed so frequently in mammalian cells, here we utilize the yeast system to examine the role of NER in the removal of AP sites in eukaryotes. Our studies indicate that Apn1, Apn2, and NER constitute alternate competing pathways for the removal of AP sites in yeast.