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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 ␤, 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 ϳ80% 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 Oxacontaining 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.

The human 3-methyladenine DNA glycosylase (AAG, MPG) removes a diverse array of damaged purines via a nucleotide-flipping mechanism. In the crystal structure of AAG bound to DNA containing 1,N(6) ethenoadenine, an asparagine (N169) occupies the active-site floor, in close proximity to the C-2 position of the flipped-out 1,N(6) ethenoadenine. We engineered site-specific AAG mutants to determine whether N169 prevents normal bases from mistakenly entering the active site. Substituting alanine or serine resulted in mutants that excised substrates at a faster rate than wild-type. Furthermore, these mutants acquired the ability to excise normal guanine within mispairs but not opposite cytosine. The results suggest that AAG can recognize helical deformations, such as mispairs. However, the active site then prevents the mistaken excision of bases, which prevents AAG from acquiring a mutator activity.

Synthetic oligonucleotides with a fluorescent coumarin group replacing a basepair have been used in recent time-resolved Stokes-shift experiments to measure DNA dynamics on the femtosecond to nanosecond timescales. Here, we show that the APE1 endonuclease cleaves such a modified oligonucleotide at the abasic site opposite the coumarin with only a fourfold reduction in rate. In addition, a noncatalytic mutant (D210N) binds tightly to the same oligonucleotide, albeit with an 85-fold reduction in binding constant relative to a native oligonucleotide containing a guanine opposite the abasic site. Thus, the modified oligonucleotide retains substantial biological activity and serves as a useful model of native DNA. In the complex of the coumarin-containing oligonucleotide and the noncatalytic APE1, the dye's absorption spectrum is shifted relative to its spectrum in either water or within the unbound oligonucleotide. Thus the dye occupies a site within the DNA:protein complex. This result is consistent with modeling, which shows that the complex accommodates coumarin at the site of the orphaned base with little distortion of the native structure. Stokes-shift measurements of the complex show surprisingly little change in the dynamics within the 40 ps-40 ns time range.

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