Abstract
Oxanine (Oxa) is a deaminated base lesion derived from guanine in which the N(1)-nitrogen is substituted by oxygen. This work reports the mutagenicity of oxanine as well as oxanine DNA glycosylase (ODG) activities in mammalian systems. Using human DNA polymerase beta, deoxyoxanosine triphosphate is only incorporated opposite cytosine (Cyt). When an oxanine base is in a DNA template, Cyt is efficiently incorporated opposite the template oxanine; however, adenine and thymine are also incorporated opposite Oxa with an efficiency approximately 80% of a Cyt/Oxa (C/O) base pair. Guanine is incorporated opposite Oxa with the least efficiency, 16% compared with cytosine. ODG activity was detected in several mammalian cell extracts. Among the known human DNA glycosylases tested, human alkyladenine glycosylase (AAG) shows ODG activity, whereas hOGG1, hNEIL1, or hNEIL2 did not. ODG activity was detected in spleen cell extracts of wild type age-matched mice, but little activity was observed in that of Aag knock-out mice, confirming that the ODG activity is intrinsic to AAG. Human AAG can excise Oxa from all four Oxa-containing double-stranded base pairs, Cyt/Oxa, Thy/Oxa, Ade/Oxa, and Gua/Oxa, with no preference to base pairing. Surprisingly, AAG can remove Oxa from single-stranded Oxa-containing DNA as well. Indeed, AAG can also remove 1,N(6)-ethenoadenine from single-stranded DNA. This study extends the deaminated base glycosylase activities of AAG to oxanine; thus, AAG is a mammalian enzyme that can act on all three purine deamination bases, hypoxanthine, xanthine, and oxanine.
Highlights
Oxanine (Oxa) is a deaminated base lesion derived from guanine in which the N1-nitrogen is substituted by oxygen
oxanine DNA glycosylase (ODG) activity was detected in spleen cell extracts of wild type age-matched mice, but little activity was observed in that of Aag knock-out mice, confirming that the ODG activity is intrinsic to alkyladenine glycosylase (AAG)
To identify enzymes that may act on oxanine in mammalian cells, we examined oxanine DNA glycosylase activities of mammalian cell extracts and of known human DNA glycosylases
Summary
5-MeC, 5-methyl cytosine; A, adenine; G, guanine; C, cytosine; I, inosine or hypoxanthine; X, xanthine; U, caused by endogenous and environmental agents [1,2,3,4]. As the deamination product of cytosine, pairs with adenine, causing Cyt to Thy transitions [13, 14]. Xanthine DNA glycosylase activities of E. coli AlkA and endo VIII proteins have been reported [9, 18]. E. coli AlkA and endo VIII contain some glycosylase activity to remove mutagenic oxanine from DNA, but it appears that a large excess of glycosylase proteins is needed to detect limited activities [18]. To understand the mutagenicity and potential oxanine DNA glycosylase activities in mammalian systems, we investigated Oxa-containing incorporation by human DNA polymerase . Human AAG can act on C/O, T/O, A/O, and G/O base pairs as well as single-stranded Oxa-containing DNA
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