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. 2008 Jan 22;47(3):1043-50.
doi: 10.1021/bi701619u. Epub 2007 Dec 23.

Efficient removal of formamidopyrimidines by 8-oxoguanine glycosylases

Nirmala Krishnamurthy  1 , Kazuhiro Haraguchi, Marc M Greenberg, Sheila S David
Affiliations

Efficient removal of formamidopyrimidines by 8-oxoguanine glycosylases

Nirmala Krishnamurthy et al. Biochemistry. .

Abstract

Under conditions of oxidative stress, the formamidopyrimidine lesions (FapyG and FapyA) are formed in competition with the corresponding 8-oxopurines (OG and OA) from a common intermediate. In order to reveal features of the repair of these lesions, and the potential contribution of repair in mitigating or exacerbating the mutagenic properties of Fapy lesions, their excision by three glycosylases, Fpg, hOGG1 and Ntg1, was examined in various base pair contexts under single-turnover conditions. FapyG was removed at least as efficiently as OG by all three glycosylases. In addition, the rates of removal of FapyG by Fpg and hOGG1 were influenced by their base pair partner, with preference for removal when base paired with the correct Watson-Crick partner C. With the FapyA lesion, Fpg and Ntg1 catalyze its removal more readily than OG opposite all four natural bases. In contrast, the removal of FapyA by hOGG1 was not as robust as FapyG or OG, and was only significant when the lesion was paired with C. The discrimination by the various glycosylases with respect to the opposing base was highly dependent on the identity of the lesion. OG induced the greatest selectivity against its removal when part of a promutagenic base pair. The superb activity of the various OG glycosylases toward removal of FapyG and FapyA in vitro suggests that these enzymes may act upon these oxidized lesions in vivo. The differences in the activity of the various glycosylases for removal of FapyG and FapyA compared to OG in nonmutagenic versus promutagenic base pair contexts may serve to alter the mutagenic profiles of these lesions in vivo.

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Figures

Figure 1
Figure 1
Formation of FapyG and OG from a common intermediate. FapyA and OA are also formed from a common intermediate in an analogous mechanism.
Figure 2
Figure 2
Sequence of the duplexes used in this study. Duplex 1 is a 36-base-pair duplex with a centrally located FapyG or OG lesion. Duplex 2 is a 30-base-pair duplex with a centrally located FapyA or OG lesion.
Figure 3
Figure 3
Representative assays for glycosylase/lyase and glycosylase actvity of hOGG1. (A) Storage phosphor autoradiogram of the removal of FapyG opposite A by hOGG1 with a dye quench to determine the glycosylase/lyase activity. Reactions were performed at 37 °C, with a DNA concentration of 20 nM and a hOGG1 concentration of 100 nM (active site concentration). The first lane is a control without the enzyme, and the triangle indicates an increase in incubation time of up to 60 min. “S” and “P” refer to the bands arising from the substrate and product duplex, respectively. (B) Storage phosphor autoradiogram of the removal of FapyG opposite A by hOGG1 with a NaOH quench to determine the glycosylase activity. Reactions were performed at 37 °C, with a DNA concentration of 20 nM and a hOGG1 concentration of 100 nM (active site concentration). The first lane is a control without the enzyme, and the triangle indicates an increase in incubation time of up to 60 min. (C) Representative plot of the removal of FapyG from FapyG:A base-pair-containing duplex 1 by hOGG1 with a dye (red circles) or a NaOH quench (green inverted triangles). The graph was derived from the quantitation of the storage phosphor autoradiograms shown in panels A and B. The data were fit to a single exponential to determine kgl or kg. Rate constants from several experiments are averaged to provide the values in Table 1.
Scheme 1
Scheme 1
Minimal Kinetic Scheme Used in Analysis of Glycosylase Activity of Fpg, hOGG1 and Ntg1a a In the case of Fpg, the lyase step is fast so it is not possible to measure the lyase rate. In contrast, glycosylase and lyase steps are uncoupled in hOGG1 and Ntg1. The kg denotes the rate constant for the glycosylase step determined by quenching the reaction with a NaOH/heat treatment. The strand cleavage step is determined by quenching the reaction with a formamide dye treatment and is denoted by kgl. In the case of Fpg, kgl = kg, while with hOGG1 and Ntg1 kgl = kl
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