Biochemistry 2010, 49, 1053–1055 DOI: 10.1021/bi902161f 1053 Evidence for the Involvement of DNA Repair Enzyme NEIL1 in Nucleotide Excision Repair of (50R)- and (50S)-8,50-Cyclo-20-deoxyadenosines† Pawel Jaruga,‡,§ Yan Xiao,‡ Vladimir Vartanian, )R. Stephen Lloyd, )and Miral Dizdaroglu*,‡ ‡Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, §Department of Clinical Biochemistry, Collegium Medicum, Nicolaus Copernicus University, Bydgoszcz, Poland, and C)enter for Research on Occupational and Environmental Toxicology, Department of Molecular and Medical Genetics, Oregon Health & Science University, Portland, Oregon 97239 Received December 18, 2009; Revised Manuscript Received January 11, 2010 ABSTRACT: The DNA repair enzyme NEIL1 is a DNA glyco- sylase that is involved in the first step of base excision repair (BER) of oxidatively induced DNA damage. NEIL1 exhi- bits a strong preference for excision of 4,6-diamino- 5-formamidopyrimidine (FapyAde) and 2,6-diamino-4- hydroxy-5-formamidopyrimidine (FapyGua) from DNA with no specificity for 8-hydroxyguanine (8-OH-Gua). In this study, we report on the significant accumulation of (50R)-8,50-cyclo-20-deoxyadenosine (R-cdA) and (50S)-8,50- cyclo-20-deoxyadenosine (S-cdA) in liver DNA of neil1-/- mice that were not exposed to exogenous oxidative stress, while no accumulation of these lesions was observed in liver DNA from control or ogg1-/- mice. Significant accumula- tion of FapyGua was detected in liver DNA of both neil1-/- and ogg1-/- mice, while 8-OH-Gua accumulated in ogg1-/- only. Since R-cdA and S-cdA contain an 8,50- covalent bond between the base and sugar moieties, they cannot be repaired by BER. There is evidence that these lesions are repaired by nucleotide excision repair (NER). Since the accumulation of R-cdA and S-cdA in neil1-/- mice strongly points to the failure of their repair, these data suggest that NEIL1 is involved in NER of R-cdA and S-cdA. Further studies aimed at elucidating the mechanism of action of NEIL1 in NER are warranted. Oxidatively induced DNA damage occurs in living cells due to endogenous and exogenous sources that cause formation of oxygen-derived species, including free radicals (1). The hydroxyl radical is one of the most reactive free radicals and causes the formation of numerous lesions in DNA (2). Among these lesions, 8,50-cyclopurine 20-deoxynucleosides contain a C8-C50 covalent bond, causing distortion of the DNA helix. Both (50R)- and (50S)- diastereomers of these compounds were identified in DNA in vitro and in vivo under various conditions (3). Most oxidatively modified DNA bases are repaired by base excision repair (BER),1 a multistep process that is initi- ated by DNA glycosylases (4). In contrast, 8,50-cyclopurine †This research was supported in part by National Institutes of Health Grant DK075974 (R.S.L.). *To whom correspondence should be addressed. Phone: (301) 975- 2581. Fax: (301) 975-8505. E-mail: miral@nist.gov. 1Abbreviations: BER, base excision repair; NER, nucleotide excision repair; R-cdA, (50R)-8,50-cyclo-20-deoxyadenosine; S-cdA, (50S)-8,50- cyclo-20-deoxyadenosine; FapyAde, 4,6-diamino-5-formamidopyrimi- dine; FapyGua, 2,6-diamino-4-hydroxy-5-formamidopyrimidine; 8- OH-Gua, 8-hydroxyguanine; CSB, Cockayne syndrome complementa- tion group B protein; LC-MS/MS, liquid chromatography-tandem mass spectrometry. 20-deoxynucleosides cannot be repaired by BER and are subject to nucleotide excision repair (NER) (5, 6). In prokaryotic and eukaryotic cells, there are numerous DNA glycosylases that possess varying and sometimes overlapping substrate specificities toward oxidatively modified DNA bases (7). Escherichia coli Nei is similar in amino acid sequence to E. coli Fpg; however, its substrate specificity resembles that of E. coli Nth (endonuclease III), albeit with no sequence homology (8). Recently, homologues of Nei were discovered in mammals and called Nei-like or NEIL proteins (9-12). Both human and mouse NEIL1 proteins were found to exhibit a strong preference for excision of 4,6-diamino-5-formamido- pyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamido- pyrimidine (FapyGua) with no significant specificity for 8-hydro- xyguanine (8-OH-Gua) (9, 13, 14). This substrate specificity is distinct from that of OGG1 that efficiently excises FapyGua and 8-OH-Gua, but not FapyAde, from DNA (15, 16). Recently, significant accumulations of FapyAde and FapyGua were observed in several organs of neil1-/- mice, unequivocally proving that these lesions are in vivo substrates of NEIL1 (17). Moreover, it was reported that the Cockayne syndrome com- plementation group B protein (CSB) stimulates the repair of FapyAde and FapyGua by NEIL1 and that CSB and NEIL1 co- immunoprecipitate and colocalize in HeLa cells (18). These results demonstrated that CSB and NEIL1 cooperate in the repair of formamidopyrimidines and that a functional and physical interaction between these proteins exists. In contrast, no direct functional and physical interactions were identified between CSB and OGG1 (19). Recently, the involvement of NEIL1 in the removal of psoralen-induced interstrand DNA cross-links was reported (20). The proposed mechanism involved the excision by NEIL1 of the unhooked interstrand cross-link fragment via hydrolysis of the glycosidic bond of the cross-linked base. Diminution or loss of NEIL1 leads to significant biological consequences. A correlation of inactivating mutations in neil1 with human gastric cancer was demonstrated (21). Knockdown of neil1 was shown to hypersensitize murine cells to killing effects of ionizing radiation (22). Downregulation of NEIL1 increased the level of spontaneous DNA damage and mutations in both Chinese hamster and human bronchial cell lines (23). Moreover, symptoms of metabolic syndrome and several types of cancer were observed in neil1-/- mice (17, 24). Collectively, the existing data strongly suggest that NEIL1 with its very distinct substrate specificity and biological properties plays a critical role in the overall main- tenance of genomic integrity and in disease prevention. r 2010 American Chemical Society Published on Web 01/12/2010 pubs.acs.org/Biochemistry