Triplet repeat sequences, such as CAG/CTG, expand in the human genome to cause several neurological disorders. As part of the expansion process the formation of non-B DNA conformations by the repeat sequence has previously been proposed. Furthermore, the base excision repair enzyme 7,8-dihydro-8-oxoguanine glycosylase (OGG1) has recently been implicated in the repeat expansion [Kovtun, I. V., Liu, Y., Bjoras, M., Klugland, A., Wilson, S. H., and McMurray, C. T. (2007) Nature 447, 447-452]. In this work we have found that the non-B conformation adopted by (CAG)(10), a hairpin, is hypersusceptible to DNA damage relative to the (CAG)(10)/(CTG)(10) duplex and, in particular, that a hot spot for DNA damage exists. Specifically, we find that a single guanine in the loop of the hairpin is susceptible to modification by peroxynitrite. Interestingly, we find that human OGG1 (hOGG1) is able to excise 7,8-dihydro-8-oxoguanine (8-oxoG) from the loop of a hairpin substrate, albeit with a marked decrease in efficiency relative to duplex substrates; the hOGG1 enzyme removes 8-oxoG from the loop of a hairpin with a rate that is approximately 700-fold slower than that observed for DNA duplex. Thus, while damage is preferentially generated in the loop of the hairpin, DNA repair is less efficient. These observed structure-dependent patterns of DNA damage and repair may contribute to the OGG1-dependent mechanism of trinucleotide repeat expansion.

Of the four native nucleosides, 2′-deoxyguanosine (dGuo) is most easily oxidized. Two lesions derived from dGuo are 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy)·dGuo. Furthermore, while steady-state levels of 8-oxodGuo can be detected in genomic DNA, it is also known that 8-oxodGuo is more easily oxidized than dGuo. Thus, 8-oxodGuo is susceptible to further oxidation to form several hyperoxidized dGuo products. This review addresses the structural impact, the mutagenic and genotoxic potential, and biological implications of oxidatively damaged DNA, in particular 8-oxodGuo, Fapy·dGuo, and the hyperoxidized dGuo products.

The repair protein 8-oxo-7,8-dihydroguanine glycosylase (OGG1) initiates base excision repair (BER) in mammalian cells by removing the oxidized base 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Interestingly, OGG1 has been implicated in somatic expansion of the trinucleotide repeat (TNR) sequence CAG/CTG. Furthermore, a ‘toxic oxidation cycle’ has been proposed for age-dependent expansion in somatic cells. In this cycle, duplex TNR DNA is (1) oxidized by endogenous species; (2) BER is initiated by OGG1 and the DNA is further processed by AP endonuclease 1 (APE1); (3) a stem-loop hairpin forms during strand-displacement synthesis by polymerase β (pol β); (4) the hairpin is ligated and (5) incorporated into duplex DNA to generate an expanded CAG/CTG region. This expanded region is again subject to oxidation and the cycle continues. We reported previously that the hairpin adopted by TNR repeats contains a hot spot for oxidation. This finding prompted us to examine the possibility that the generation of a hairpin during a BER event exacerbates the toxic oxidation cycle due to accumulation of damage. Therefore, in this work we used mixed-sequence and TNR substrates containing a site-specific 8-oxoG lesion to define the kinetic parameters of human OGG1 (hOGG1) activity on duplex and hairpin substrates. We report that hOGG1 activity on TNR duplexes is indistinguishable from a mixed-sequence control. Thus, BER is initiated on TNR sequences as readily as non-repetitive DNA in order to start the toxic oxidation cycle. However, we find that for hairpin substrates hOGG1 has reduced affinity and excises 8-oxoG at a significantly slower rate as compared to duplexes. Therefore, 8-oxoG is expected to accumulate in the hairpin intermediate. This damage-containing hairpin can then be incorporated into duplex, resulting in an expanded TNR tract that now contains an oxidative lesion. Thus, the cycle restarts and the DNA can incrementally expand.