1054 Biochemistry, Vol. 49, No. 6, 2010 Scheme 1: Structures of R-cdA and S-cdA Jaruga et al. Thus far, the accumulated evidence unequivocally shows that NEIL1 is a DNA glycosylase and may also be preferentially active in transcription- and/or replication-coupled repair of DNA base lesions and interstrand cross-links. No additional role for NEIL1 in DNA repair mechanisms was demonstrated. Recently, neil1-/- mice were shown to exhibit the symptoms of metabolic syndrome and to develop several types of cancer despite the lack of exogenous oxidative stress (17, 24). The accumulation of FapyAde and FapyGua, but not 8-OH-Gua, was observed in these mice. In this work, we hypothesize that NEIL1 may be involved in the repair of more complex DNA lesions than those resulting from modifications of DNA bases only. A recent study showed the accumulation of (50S)-8,50-cyclo- 20-deoxyadenosine (S-cdA) in several organs of csb-/- mice without exogenous oxidative stress, suggesting a critical role of CSB in the in vivo NER of this lesion (25). The aforementioned cooperation of CSB and NEIL1 in the repair of FapyAde and FapyGua may also be true for other more complex lesions. We tested this hypothesis by analyzing nuclear DNA samples from livers of neil1-/- mice and wild-type mice for R-cdA and S-cdA. We also generated ogg1-/- mice and tested their liver nuclear DNA as well for comparison. The structures of R-cdA and S-cdA are shown in Scheme 1. We utilized a methodology that we had recently developed with the use of liquid chromatography and isotope-dilution tandem mass spectrometry (LC-MS/MS) (26). This technique is capable of the positive identification and accurate quantification of very low endogenous levels of both R-cdA and S-cdA. Mouse liver samples were blinded and shipped on ice. Nuclear DNA was isolated from unfrozen livers imme- diately upon arrival. Figure 1 illustrates the ion-current profiles of m/z 250 f m/z 164 (cdA) and m/z 255 f m/z 169 (cdA-15N5) transitions obtained during analysis of a nuclear DNA sample from a neil1-/- mouse liver. The levels of S-cdA in the livers of wild- type, neil1-/-, and ogg1-/- mice are shown in Figure 2. These data unequivocally showed the statistically significant accumula- tion of S-cdA in neil1-/- mice. In contrast, no accumulation of S-cdA was observed in ogg1-/- mice. R-cdA was quantified in neil1-/- mice only because of its insignificant levels in wild-type and ogg1-/- mice. Its measured level was 0.041 ( 0.013 molecule/ 106 DNA bases. Thus, we concluded that R-cdA also significantly accumulated in neil1-/- mice. In addition, we measured the levels of FapyGua and 8-OH-Gua using GC-MS. Figures 3 and 4 illustrate the levels of FapyGua and 8-OH-Gua in liver DNA of wild-type, neil1-/-, and ogg1-/- mice, respec- tively. A statistically significant accumulation of FapyGua was observed in neil1-/- and ogg1-/- mice. These data confirm our previous findings (17, 18) and are on par with the substrate specificities of NEIL1 and OGG1 determined under in vitro conditions (9, 13-16). In contrast, 8-OH-Gua accumulated in ogg1-/- mice only (Figure 4), in agreement with the known specificity of OGG1 for 8-OH-Gua. As expected from the FIGURE 1: Ion-current profiles of the m/z 250 f m/z 164 (cdA) and m/z 255 f m/z 169 (cdA-15N5) transitions. FIGURE 2: Levels of S-cdA in liver DNA of wild-type (1) (n = 4), neil1-/- (2) (n = 7), and ogg1-/- (3) (n = 9) mice. The uncertainties are standard deviations. *p < 0.05. FIGURE 3: Levels of FapyGua in liver DNA of wild-type (1) (n = 4), neil1-/- (2) (n = 6), and ogg1-/- (3) (n = 7) mice. The uncertainties are standard deviations. *p < 0.05. substrate specificity of NEIL1, no accumulation of 8-OH-Gua was observed in neil1-/- mice, in agreement with our previous results (17). This study strongly suggests that NEIL1 plays a role in the cellular repair of R-cdA and S-cdA. Since these compounds are repaired by NER and not by BER, these results suggest for the first time that NEIL1 is involved in NER in addition to its function as a DNA glycosylase in BER. A previous study demonstrated that CSB stimulates the repair of formamido- pyrimidines by NEIL1. Moreover, CSB was shown to play a role in NER of S-cdA. Thus, in turn, NEIL1 may be essential for the involvement of CSB in NER of R-cdA and S-cdA. On the other