Repair of Oxidative Damage to Nuclear and Mitochondrial DNA in Mammalian Cells

Abstract

Reactive oxygen species (ROS) 1The abbreviations used are: ROS, reactive oxygen species; BER, base excision repair; NER, nucleotide excision repair; AD, Alzheimer's disease; endo, endonuclease; TCR, transcription-coupled repair; 8-oxoG, 8-hydroxyguanine; 8-oxodG, 8-hydroxydeoxyguanine; TG, thymine glycol; AP, apurinic/apyrimidinc; GSR, gene-specific repair assay; XP, xeroderma pigmentosum; CS, Cockayne's syndrome; FapyG, 2,6-diamino-4hydroxyl-5-methylformamidopyrimidine. 1The abbreviations used are: ROS, reactive oxygen species; BER, base excision repair; NER, nucleotide excision repair; AD, Alzheimer's disease; endo, endonuclease; TCR, transcription-coupled repair; 8-oxoG, 8-hydroxyguanine; 8-oxodG, 8-hydroxydeoxyguanine; TG, thymine glycol; AP, apurinic/apyrimidinc; GSR, gene-specific repair assay; XP, xeroderma pigmentosum; CS, Cockayne's syndrome; FapyG, 2,6-diamino-4hydroxyl-5-methylformamidopyrimidine. are generated in cells as a by-product of cellular metabolism. ROS react with proteins, lipids, and DNA. DNA base modifications, abasic sites, deoxyribose damage, and single and double strand breaks are all induced following various forms of oxidative stress. This review will focus on DNA repair of oxidative lesions by base excision repair (BER) and nucleotide excision repair (NER). We will focus on the mammalian BER enzymes that have recently been cloned and characterized. Mitochondrial DNA repair mechanisms for oxidative damage will also be discussed. Although sugar damage and double strand breaks are critical lesions induced by ionizing radiation and bleomycin, repair of these lesions will not be discussed here (see Refs. 1Weaver D.T. Crit. Rev. Eukaryotic Gene Expression. 1996; 6: 345-375Crossref PubMed Scopus (44) Google Scholar, 2Lieber M.R. Grawunder U. Wu X. Yaneva M. Curr. Opin. Genet. Dev. 1997; 7: 99-104Crossref PubMed Scopus (127) Google Scholar, 3Povirk L.F. Mutat. Res. 1996; 355: 71-89Crossref PubMed Scopus (333) Google Scholar for recent reviews).Oxidative DNA Damage and Its ConsequencesThe endogenous attack on DNA by ROS species generates a low steady-state level of DNA adducts that have been detected in the DNA from human cells (4Dizdaroglu M. Halliwell B. Aruoma O.I. DNA and Free Radicals. Ellis Horwood, Ltd., London, United Kingdom1993: 18-39Google Scholar). Some of these base modifications are shown in Fig. 1. There are many more, and it is possible that the full spectrum of oxidative lesions in endogenous mammalian DNA exceeds 100 different types, of which 8-hydroxyguanine (8-oxoG) is one of the most abundant (5Ames B.N. Free Radical Res. Commun. 1989; 7: 121-128Crossref PubMed Scopus (627) Google Scholar).Oxidative DNA damage is thought to contribute to carcinogenesis, aging, and neurological degeneration (for reviews, see Refs. 5Ames B.N. Free Radical Res. Commun. 1989; 7: 121-128Crossref PubMed Scopus (627) Google Scholar and 6Wiseman H. Halliwell B. Biochem. J. 1996; 313: 17-29Crossref PubMed Scopus (1943) Google Scholar). Studies have shown that oxidative DNA damage accumulates in cancerous tissue. For example, higher levels of oxidative base damage were observed in lung cancer tissue compared with surrounding normal tissue (7Olinski R. Zastawny T. Budzbin J. Skokowski J. Zegarski W. Dizdaroglu M. FEBS Lett. 1992; 309: 193-198Crossref PubMed Scopus (243) Google Scholar). Another study reported a 9-fold increase in 8-oxoG, 8-hydroxyadenine, and 2,6-diamino-4-hydroxy-5-formamidopyrimidine in DNA from breast cancer tissue compared with normal tissue (8Malins D.C. Haimanot R. Cancer Res. 1991; 51: 5430-5432PubMed Google Scholar). Further, the cumulative risk of cancer increases dramatically with age in humans (9Ames B.N. Mutat. Res. 1989; 214: 41-46Crossref PubMed Scopus (290) Google Scholar), and cancer can in general terms be regarded as a degenerative disease of old age. There is evidence for the accumulation of oxidative DNA damage with age based on studies mainly measuring the increase in 8-oxoG (10Sohal R.S. Ku H.H. Agarwal S. Forster M.J. Lal H. Mech. Ageing Dev. 1994; 74: 121-133Crossref PubMed Scopus (689) Google Scholar). In Alzheimer's disease (AD), some studies have shown an accumulation of oxidative DNA damage in the brain, and a recent extensive study in cells from familial Alzheimer's disease demonstrated a deficiency in the processing of damage invoked by fluorescent light (11Parshad R.P. Sanford K.K. Price F.M. Melnick L.K. Nee L.E. Schapiro M.B. Tarone R.E. Robbins J.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5146-5150Crossref PubMed Scopus (62) Google Scholar). The effects of fluorescent light exposure were inhibited by the addition of free radical scavengers, and therefore it was proposed that oxidative DNA damage was produced and responsible for the altered response seen in AD cells (11Parshad R.P. Sanford K.K. Price F.M. Melnick L.K. Nee L.E. Schapiro M.B. Tarone R.E. Robbins J.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5146-5150Crossref PubMed Scopus (62) Google Scholar). AD cells also respond abnormally to ionizing radiation and simple alkylating agents, and therefore it is possible that lesions introduced by these agents such as oxidative modifications, alkylpurines, or DNA strand breaks are not repaired efficiently in AD cells (12Scudiero D.A. Polinsky R.J. Brumbach R.A. Tarone R.E. Nee L.E. Robbins J.H. Mutat. Res. 1986; 159: 125-131Crossref PubMed Scopus (45) Google Scholar).Many experimental methods have been used to expose cells to oxidative damage, all attempting to mimic endogenous processes (4Dizdaroglu M. Halliwell B. Aruoma O.I. DNA and Free Radicals. Ellis Horwood, Ltd., London, United Kingdom1993: 18-39Google Scholar, 6Wiseman H. Halliwell B. Biochem. J. 1996; 313: 17-29Crossref PubMed Scopus (1943) Google Scholar). Some studies have used hydrogen peroxide, which generates a large spectrum of lesions. Ionizing radiation also generates a wide spectrum of lesions including base damage and single and double strand breaks in DNA. Methylene blue plus visible light exposure primarily generates singlet oxygen damage, and osmium tetroxide generates primarily thymine glycols. For more discussion of this see Refs. 4Dizdaroglu M. Halliwell B. Aruoma O.I. DNA and Free Radicals. Ellis Horwood, Ltd., London, United Kingdom1993: 18-39Google Scholar and6Wiseman H. Halliwell B. Biochem. J. 1996; 313: 17-29Crossref PubMed Scopus (1943) Google Scholar. It is important to distinguish between the different types of oxidative stresses when evaluating experimental results.Technical differences in the methods used for DNA isolation may well result in differences in the analysis of the DNA adducts. A recent review compared the various methods used to detect oxidative damage in DNA (13Beckman K.B. Ames B.N. Methods Enzymol. 1996; 264: 442-453Crossref PubMed Google Scholar). One of the conclusions that emerged from the comparison was that there is a great need for methods to be more standardized and thus to provide more consistent results between different laboratories when comparing different but related techniques.One aspect that is common to many methods used to detect oxidative damage is that the DNA modifications are measured as averages in the total cellular DNA. This is of limited value since advances in recent years have shown that DNA damage processing and the biological consequences of DNA lesions vary considerably depending upon where a lesion is situated in the genome. For example, UV-induced photoproducts are processed differently whether situated in an active gene or in a non-transcribed region, and this may also be the case for oxidative lesions.The gene-specific repair assay (GSR) employs various DNA repair enzymes to detect specific lesions, and this assay has provided new insights about the heterogeneity of DNA repair in the nucleus (14Bohr V.A. Carcinogenesis. 1995; 16: 2885-2892Crossref PubMed Scopus (95) Google Scholar) and more recently about the repair mechanisms for mitochondrial DNA (see below). For example, endonuclease III (endo III) can detect oxidized pyrimidines, and the Fapy DNA glycosylase (Fpg protein) can detect oxidized purines. Endo III-sensitive sites have been assayed in the general genome (15Collins A.R. Duthie S.J. Dobson V.L. Carcinogenesis. 1993; 14: 1733-1735Crossref PubMed Scopus (761) Google Scholar), and more recently, Fpg protein has been used to detect lesions in specific genes (16Driggers W.J. LeDoux S.P. Wilson G.L. J. Biol. Chem. 1993; 268: 22042-22045Abstract Full Text PDF PubMed Google Scholar, 17Taffe B.G. Larminat F. Laval J. Croteau D.L. Anson R.M. Bohr V.A. Mutat. Res. 1996; 364: 183-192Crossref PubMed Scopus (64) Google Scholar).Base Excision Repair of Oxidative DamageBER is initiated by DNA glycosylases, a class of enzymes that recognize a specific set of modified bases such as 8-oxoG or thymine glycol (TG). Glycosylases cleave the N-glycosylic bond between the modified base and the sugar. There are two classifications of glycosylases: simple glycosylases that only cleave the N-glycosylic bond and glycosylase/AP lyase enzymes, which cleave the N-glycosylic bond and the DNA-phosphate backbone. Following the glycosylase step, AP endonucleases are required to remove the 3′-deoxyribose moiety and generate a 3′-hydroxyl group, which can be extended by a DNA polymerase. The process is completed by a DNA ligase rejoining the free DNA ends (for reviews see Refs. 18Seeberg E. Eide L. Bjoras M. Trends Biochem. Sci. 1995; 20: 391-397Abstract Full Text PDF PubMed Scopus (465) Google Scholar and19Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. American Society for Microbiology, Wash., D. C.1995: 135-190Google Scholar).Repair of 8-oxoGThe majority of our knowledge regarding the repair of 8-oxoG has been derived from studies in Escherichia coli. 8-oxoG is considered to be a premutagenic lesion because it can mispair with adenine during DNA replication, and this mispairing results in G → T transversion mutations (20Grollman A.P. Moriya M. Trends Genet. 1993; 9: 246-249Abstract Full Text PDF PubMed Scopus (727) Google Scholar). Bacteria possess an integrated system of BER and error avoidance mechanisms to prevent damage at guanines (for a review see Ref. 20Grollman A.P. Moriya M. Trends Genet. 1993; 9: 246-249Abstract Full Text PDF PubMed Scopus (727) Google Scholar). This system is comprised of three components, an 8-oxoG glycosylase/AP lyase enzyme, called MutM or Fpg protein, an adenine DNA glycosylase, MutY, and a 8-oxodGTPase, MutT. As will be discussed, functional homologs of each of these proteins have now been identified in higher eukaryotes.Two groups have independently cloned an 8-oxoguanine glycosylase/AP lyase from yeast (yOgg1) (21van der Kemp P.A. Thomas D. Barbey R. de Oliveira R. Boiteux S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5197-5202Crossref PubMed Scopus (346) Google Scholar, 22Nash H.M. Bruner S.D. Schaerer O.D. Kawate T. Addona T.A. Spooner E. Lane W.S. Verdine G.L. Curr. Biol. 1996; 6: 968-980Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar). The enzyme is a functional homolog of the Fpg protein because the yeast enzyme shares no amino acid homology with the bacterial protein. The yOgg1 cleaved DNA containing 8-oxoG opposite pyrimidines, abasic sites (21van der Kemp P.A. Thomas D. Barbey R. de Oliveira R. Boiteux S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5197-5202Crossref PubMed Scopus (346) Google Scholar, 22Nash H.M. Bruner S.D. Schaerer O.D. Kawate T. Addona T.A. Spooner E. Lane W.S. Verdine G.L. Curr. Biol. 1996; 6: 968-980Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar), and 2,6-diamino-4-hydroxy-5-methylformamidopyrimidine (FapyG) (21van der Kemp P.A. Thomas D. Barbey R. de Oliveira R. Boiteux S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5197-5202Crossref PubMed Scopus (346) Google Scholar). Cleavage by yOgg1 was consistent with a β-elimination mechanism (21van der Kemp P.A. Thomas D. Barbey R. de Oliveira R. Boiteux S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5197-5202Crossref PubMed Scopus (346) Google Scholar,22Nash H.M. Bruner S.D. Schaerer O.D. Kawate T. Addona T.A. Spooner E. Lane W.S. Verdine G.L. Curr. Biol. 1996; 6: 968-980Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar).Recently, the human and the mouse 8-oxoguanine glycosylase/AP lyase (human OGG1 or mouse Ogg1) genes have been cloned by their homology to yeast ogg1 (23Lu R. Nash H.M. Verdine G.L. Curr. Biol. 1997; 7: 397-407Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 24Aburatani H. Hippo Y. Ishida T. Takashima R. Matsuba C. Kodama T. Takao M. Yasui A. Yamamoto K. Asano M. Fukasawa K. Yoshinari T. Inoue H. Ohtsuka E. Nishimura S. Cancer Res. 1997; 57: 2151-2156PubMed Google Scholar, 25Arai K. Morishita K. Shinmura K. Kohno T. Kim S. Nohmi T. Taniwaki M. Ohwada S. Yokota J. Oncogene. 1997; 14: 2857-2861Crossref PubMed Scopus (247) Google Scholar). Human OGG1 gene was localized to the short arm of chromosome 3, 3p26.2 (23Lu R. Nash H.M. Verdine G.L. Curr. Biol. 1997; 7: 397-407Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 25Arai K. Morishita K. Shinmura K. Kohno T. Kim S. Nohmi T. Taniwaki M. Ohwada S. Yokota J. Oncogene. 1997; 14: 2857-2861Crossref PubMed Scopus (247) Google Scholar). Expression of the human gene in E. coli lacking mutM and mutY suppressed the spontaneous mutator phenotype of these cells (24Aburatani H. Hippo Y. Ishida T. Takashima R. Matsuba C. Kodama T. Takao M. Yasui A. Yamamoto K. Asano M. Fukasawa K. Yoshinari T. Inoue H. Ohtsuka E. Nishimura S. Cancer Res. 1997; 57: 2151-2156PubMed Google Scholar, 25Arai K. Morishita K. Shinmura K. Kohno T. Kim S. Nohmi T. Taniwaki M. Ohwada S. Yokota J. Oncogene. 1997; 14: 2857-2861Crossref PubMed Scopus (247) Google Scholar). Human OGG1 (also called MutM homolog) was shown to cleave the DNA by a β-elimination mechanism preferentially at 8-oxoG:C base pairs (23Lu R. Nash H.M. Verdine G.L. Curr. Biol. 1997; 7: 397-407Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 24Aburatani H. Hippo Y. Ishida T. Takashima R. Matsuba C. Kodama T. Takao M. Yasui A. Yamamoto K. Asano M. Fukasawa K. Yoshinari T. Inoue H. Ohtsuka E. Nishimura S. Cancer Res. 1997; 57: 2151-2156PubMed Google Scholar). Several conserved domains have been identified in the yeast, mouse, and human genes including the a helix-hairpin-helix (HhH) and Gly/Pro-rich-Asp motif (GPD motif) (22Nash H.M. Bruner S.D. Schaerer O.D. Kawate T. Addona T.A. Spooner E. Lane W.S. Verdine G.L. Curr. Biol. 1996; 6: 968-980Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar, 23Lu R. Nash H.M. Verdine G.L. Curr. Biol. 1997; 7: 397-407Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 25Arai K. Morishita K. Shinmura K. Kohno T. Kim S. Nohmi T. Taniwaki M. Ohwada S. Yokota J. Oncogene. 1997; 14: 2857-2861Crossref PubMed Scopus (247) Google Scholar). In addition, Arai et al. (25Arai K. Morishita K. Shinmura K. Kohno T. Kim S. Nohmi T. Taniwaki M. Ohwada S. Yokota J. Oncogene. 1997; 14: 2857-2861Crossref PubMed Scopus (247) Google Scholar) reported that the yeast Ogg1 and human OGG1 contained a putative C2H2 zinc finger-like motif, although in the yeast sequence one of the histidines was an arginine. Alignment of the ogg1 genes with other DNA repair glycosylases suggests that these enzymes may represent a DNA repair superfamily (18Seeberg E. Eide L. Bjoras M. Trends Biochem. Sci. 1995; 20: 391-397Abstract Full Text PDF PubMed Scopus (465) Google Scholar, 22Nash H.M. Bruner S.D. Schaerer O.D. Kawate T. Addona T.A. Spooner E. Lane W.S. Verdine G.L. Curr. Biol. 1996; 6: 968-980Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar, 23Lu R. Nash H.M. Verdine G.L. Curr. Biol. 1997; 7: 397-407Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 25Arai K. Morishita K. Shinmura K. Kohno T. Kim S. Nohmi T. Taniwaki M. Ohwada S. Yokota J. Oncogene. 1997; 14: 2857-2861Crossref PubMed Scopus (247) Google Scholar).Nash et al. (22Nash H.M. Bruner S.D. Schaerer O.D. Kawate T. Addona T.A. Spooner E. Lane W.S. Verdine G.L. Curr. Biol. 1996; 6: 968-980Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar) identified another yeast protein, which preferentially interacted with the substrate 8-oxoG:G; they called the activity Ogg2. This same substrate preference was observed for the yeast Fapy DNA glycosylase previously isolated by de Oliveira et al. (26de Oliveira R. van der Kemp P.A. Thomas D. Geiger A. Nehls P. Boiteux S. Nucleic Acids Res. 1994; 22: 3760-3764Crossref PubMed Scopus (35) Google Scholar). Whether these two proteins are the same or not remains to be determined. In human extracts, an 8-oxoG endonuclease was identified from human polymorphonuclear neutrophils, which cleaved 8-oxoG but not the ring-opened guanine adduct, FapyG (27Chung M.H. Kim H.S. Ohtsuka E. Kasai H. Yamamoto F. Nishimura S. Biochem. Biophys. Res. Commun. 1991; 178: 1472-1478Crossref PubMed Scopus (84) Google Scholar). One distinguishing feature of this enzyme was that it was magnesium-dependent. Another study identified two repair activities, an 8-oxoG glycosylase and an 8-oxoG endonuclease, from HeLa cell nuclear extracts (28Bessho T. Tano K. Kasai H. Ohtsuka E. Nishimura S. J. Biol. Chem. 1993; 268: 19416-19421Abstract Full Text PDF PubMed Google Scholar). The 8-oxoG base pairing preferences for these enzymes were similar to that of yeast Ogg1. Further experiments are required to determine whether these proteins are human OGG1 or novel enzymes.In E. coli, the MutY protein is an adenine DNA glycosylase that removes adenine when base paired with 8-oxoG. Using purified DNA polymerases, it has been demonstrated that the replicative polymerases incorporate adenine opposite 8-oxoG (29Shibutani S. Takeshita M. Grollman A.P. Nature. 1991; 349: 431-434Crossref PubMed Scopus (2024) Google Scholar). A human MutY activity has been purified from calf thymus cells (30McGoldrick J.P. Yeh Y.C. Solomon M. Essigmann J.M. Lu A.L. Mol. Cell. Biol. 1995; 15: 989-996Crossref PubMed Google Scholar). The protein removes adenine mispairs including A:G, A/8-oxoG, and A:C. The glycosylase co-purified with a AP nicking activity, which was inhibited by neutralizing MutY antibodies. Recently, the gene for a human MutY homolog was cloned (31Slupska M.M. Baikalov C. Luther W.M. Chiang J.H. Wei Y.F. Miller J.H. J. Bacteriol. 1996; 178: 3885-3892Crossref PubMed Scopus (327) Google Scholar).In cells, the deoxyribonucleotide pools are also subjected to oxidative damage. dGTP can be converted to 8-oxodGTP and incorporated into nascent DNA strands opposite adenine. To avoid such damage, cells possess an 8-oxodGTPase, which hydrolyzes the triphosphate to the monophosphate so that it can no longer be incorporated into DNA. In bacteria, the MutT gene product is the 8-oxodGTPase enzyme. A human MutT homolog has been cloned from a human cell line (32Sakumi K. Furuichi M. Tsuzuki T. Kakuma T. Kawabata S. Maki H. Sekiguchi M. J. Biol. Chem. 1993; 268: 23524-23530Abstract Full Text PDF PubMed Google Scholar).Repair of Thymine Glycols and Ring-saturated PyrimidinesAnother major adduct generated by oxidative stress is TGs (cf. Fig. 1). Unlike 8-oxoG, TGs block DNA and RNA polymerases and are thought to be lethal (33Ide H. Tedzuka K. Shimzu H. Kimura Y. Purmal A.A. Wallace S.S. Kow Y.W. Biochemistry. 1994; 33: 7842-7847Crossref PubMed Scopus (95) Google Scholar). Endo III is one of the bacterial enzymes responsible for recognition and removal of TGs; however, cells lacking endo III are not hypersensitive to H2O2 or x-rays (34Cunningham R.P. Weiss B. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 474-478Crossref PubMed Scopus (145) Google Scholar). Subsequently it was shown that bacteria contain another endonuclease that recognizes TG, endonuclease VIII (35Melamede R.J. Hatahet Z. Kow Y.W. Ide H. Wallace S.S. Biochemistry. 1994; 33: 1255-1264Crossref PubMed Scopus (160) Google Scholar). In addition, the Uvr ABC complex was shown to recognize TGs in vitro (36Kow Y.W. Wallace S.S. Van Houten B. Mutat. Res. 1990; 235: 147-156Crossref PubMed Scopus (103) Google Scholar). Recently, a yeast homolog of endo III has been cloned, NTG1 (endonucleasethree-like glycosylase 1) (37Eide L. Bjoras M. Pirovano M. Alseth I. Berdal K.G. Seeberg E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10735-10740Crossref PubMed Scopus (146) Google Scholar). NTG1 has a unique substrate specificity; not only does it remove oxidized purines, but it also recognizes and incises the ring-opened guanine adduct, FapyG. However, it does not incise the 8-oxodG adduct (37Eide L. Bjoras M. Pirovano M. Alseth I. Berdal K.G. Seeberg E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10735-10740Crossref PubMed Scopus (146) Google Scholar). Deletion of yeast NTG1 renders the cells sensitive to H2O2 and menadione (37Eide L. Bjoras M. Pirovano M. Alseth I. Berdal K.G. Seeberg E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10735-10740Crossref PubMed Scopus (146) Google Scholar).A mammalian TG glycosylase activity has been purified from extracts of calf thymus and bovine cells (38Hilbert T.P. Boorstein R.J. Kung H.C. Bolton P.H. Xing D. Cunningham R.P. Teebor G.W. Biochemistry. 1996; 35: 2505-2511Crossref PubMed Scopus (78) Google Scholar). More recently another gene for the human endonuclease III homolog was cloned (39Aspinwall R. Rothwell D.G. Roldan-Arjona T. Anselmino C. Ward C.J. Cheadle J.P. Sampson J.R. Lindahl T. Harris P.C. Hickson I.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 109-114Crossref PubMed Scopus (254) Google Scholar). Like the bacterial enzyme endo III, the human enzyme acts on urea and TG residues. It also contains an iron/sulfur cluster and a helix-hairpin-helix motif.Repair of Abasic Sites and Sugar DamageApurinic or apyrimidinic sites (AP sites) are collectively called abasic sites. These are generated as a consequence of normal spontaneous hydrolysis of the N-glycosylic bond, by the action of DNA glycosylases or by oxidative damage to the sugar residues in DNA. AP endonucleases are enzymes that function to generate suitable DNA ends for DNA resynthesis or ligation (for reviews see Refs. 40Doetsch P.W. Cunningham R.P. Mutat. Res. 1990; 236: 173-201Crossref PubMed Scopus (322) Google Scholar, 41Demple B. Harrison L. Annu. Rev. Biochem. 1994; 63: 915-948Crossref PubMed Scopus (1287) Google Scholar, 42Barzilay G. Hickson I.D. BioEssays. 1995; 17: 713-719Crossref PubMed Scopus (196) Google Scholar). One major AP endonuclease has been purified from human cells called HAP1 (also called APE, APEX, and Ref-1) (reviewed in Ref. 42Barzilay G. Hickson I.D. BioEssays. 1995; 17: 713-719Crossref PubMed Scopus (196) Google Scholar). The enzyme cleaves 5′ to the AP site leaving a 5′-deoxyribose moiety and a 3′-hydroxyl group on the DNA ends. In addition to the AP endonuclease activity, the enzyme possesses several other activities including a 3′-phosphatase, 3′-phosphodiesterase, and a very weak exonuclease activity (42Barzilay G. Hickson I.D. BioEssays. 1995; 17: 713-719Crossref PubMed Scopus (196) Google Scholar, 43Seki S. Ikeda S. Watanabe S. Hatsushika M. Tsutsui K. Akiyama K. Zhang B. Biochim. Biophys. Acta. 1991; 1079: 57-64Crossref PubMed Scopus (75) Google Scholar, 44Wilson III, D.M. Takeshita M. Grollman A.P. Demple B. J. Biol. Chem. 1995; 270: 16002-16007Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar).AP endonucleases are the major pathway whereby AP sites in DNA are repaired; however, nucleotide excision may also participate (42Barzilay G. Hickson I.D. BioEssays. 1995; 17: 713-719Crossref PubMed Scopus (196) Google Scholar). In E. coli, oxidized abasic sites are poorly recognized by various repair endonucleases (45Haring M. Rudiger H. Demple B. Boiteux S. Epe B. Nucleic Acids Res. 1994; 22: 2010-2015Crossref PubMed Scopus (100) Google Scholar). It would be interesting to know whether the mammalian HAP1 also displays this reduced recognition and incision of oxidized AP sites. If so, then what other pathways participate in the repair of oxidized AP sites? Do NER proteins recognize and repair oxidized AP sites?It is already apparent that repair of oxidative damage by a BER mechanism in mammalian cells is more complex than in bacteria. Due to the high levels of endogenous oxidative damage, mammalian cells may have had to evolve multiple repair mechanisms to survive the daily insults. Therefore, it may be difficult to define what significance a particular protein has on the repair of specific types of DNA damage, and generation of single-gene knock-out mice may not be informative. Multiple single-gene knock-out mice may have to be crossbred before a phenotype is observed.Nucleotide Excision Repair of Oxidative DamageIn bacteria and mammalian cells, the repair of oxidative damage is mediated by both BER and NER mechanisms (46Satoh M.S. Jones C.J. Wood R.D. Lindahl T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6335-6339Crossref PubMed Scopus (161) Google Scholar, 47Lindahl T. Nature. 1993; 362: 709-715Crossref PubMed Scopus (4273) Google Scholar). NER employs a complex set of proteins that remove damage from DNA (recently reviewed in Refs.48Wood R.D. Annu. Rev. Biochem. 1996; 65: 135-167Crossref PubMed Scopus (611) Google Scholar and 49Sancar A. Annu. Rev. Biochem. 1996; 65: 43-81Crossref PubMed Scopus (960) Google Scholar). There are two components of NER, a global repair element and a TCR mechanism. Transcriptionally active genes are repaired at a faster rate than genes in non-transcriptionally active domains of the genome and with a strand bias favoring the transcribed DNA strand (50Friedberg E.C. Annu. Rev. Biochem. 1996; 65: 15-42Crossref PubMed Scopus (212) Google Scholar). The coupling between DNA repair and transcription is mediated via the basal transcription factor, TFIIH, which contains at least two DNA repair genes (50Friedberg E.C. Annu. Rev. Biochem. 1996; 65: 15-42Crossref PubMed Scopus (212) Google Scholar). Three genetic disorders have been identified that have defective NER, xeroderma pigmentosum (XP), Cockayne's syndrome (CS), and trichothiodystrophy.Patients with XP are characterized by their acute sun sensitivity and the development of carcinomas at an early age. Seven complementation groups of XP have been identified, and several XP and XP/CS groups develop neurological abnormalities. Studies were performed to determine whether the oxidative damage repair capacity of the XP cells correlated with their neurological phenotypes (51Runger T.M. Epe B. Moller K. J. Invest. Dermatol. 1995; 104: 68-73Abstract Full Text PDF PubMed Scopus (32) Google Scholar, 52Nocentini S. Mutat. Res. 1992; 284: 275-285Crossref PubMed Scopus (6) Google Scholar). A host cell reactivation assay was used to measure the capability of two XP-A cell lines to repair viral DNA, which had been damaged by singlet oxygen (52Nocentini S. Mutat. Res. 1992; 284: 275-285Crossref PubMed Scopus (6) Google Scholar). The XP-A cells showed no difference from normal cells. In another study, a chloramphenicol acetyltransferase reactivation assay was employed to evaluate the survival of a methylene blue plus light-treated DNA in XP-A, XP-C, XP-D, and XP-E cell lines. Using seven normal cell lines, the investigators first defined a normal response range and then determined whether the XP cell lines fell within this normal range. Of the cell lines tested, only the XP-C (3 out of 4) cell lines showed reduced chloramphenicol acetyltransferase reactivation. Whereas UV repair is compromised in all XP complementation groups to varying degrees, the repair of singlet oxygen damage is not. XP-A cells show the greatest sensitivity to UV damage, and most patients with XP-A have demonstrable neurological abnormalities. However, XP-A cells appear to be normal in their repair of singlet oxygen-mediated damage. This suggests that there is no direct relationship between the accumulation of singlet oxygen damage in DNA and the development of neurological defects.It is possible that an oxidative DNA lesion other than those generated by singlet oxygen may be critical in the development of neurological defects in XP cells. This is supported by a study which investigated whether repair of lesions other than the major oxidative adducts was defective in NER-deficient XP cell lines. Satoh et al. (46Satoh M.S. Jones C.J. Wood R.D. Lindahl T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6335-6339Crossref PubMed Scopus (161) Google Scholar) treated DNA plasmids with either γ-irradiation or hydrogen peroxide plus copper and then removed the major adducts by treating the plasmids with the Fpg protein and endo III (46Satoh M.S. Jones C.J. Wood R.D. Lindahl T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6335-6339Crossref PubMed Scopus (161) Google Scholar). They then assayed whether XP-A, XP-B, XP-C, XP-D, and XP-G cell extracts were able to perform DNA repair synthesis on such substrates. All XP cell lines showed reduced DNA repair synthesis as compared with normal cell extracts. The specific lesion in the plasmid DNA, which was dependent on NER for repair, was proposed to be purine dimers. The authors concluded that although repair of the major oxidative lesions is not impaired in XP cell extracts, there could be some endogenous oxidative lesions that are formed at low levels, require NER for repair, that accumulate in XP patients, and lead to neurological defects.CS patients are characterized by dwarfism, premature aging, sensitivity to sunlight, and mental retardation (53Friedberg E.C. BioEssays. 1996; 18: 731-738Crossref PubMed Scopus (80) Google Scholar). Patients with features of both XP and CS patients have been identified for XP groups, XP-B, XP-D, and XP-G (53Friedberg E.C. BioEssays. 1996; 18: 731-738Crossref PubMed Scopus (80) Google Scholar). CS cells are deficie