Abstract
In vitro selection was used to define sequence contexts that significantly enhanced the mutagenic potential of 7, 8-dihydro-8-oxoguanine (8-oxoG). Contexts that simultaneously reduced the efficiency of 8-oxoG cleavage by formamidopyrimidine DNA N-glycosylase and increased the efficiency of misincorporating A opposite the lesion by DNA polymerase were isolated from a pool of 4(8) random octanucleotide sequences. Kinetic analysis showed that the combined effects of poor repair and high miscoding resulted in 10(2)- to 10(3)-fold increase in the mutagenic potential of 8-oxoG. Furthermore, the isolated sequence contexts correlated strongly with G --> T transversion hotspots in spontaneous mutational spectra reported for the Escherichia coli lacI and human p53 and factor IX genes. We present an example directly linking the interplay between DNA repair and replication to a "high risk sequence" for base substitution.
Highlights
DNA damage-induced mutational spectra obtained from a wide range of species often display regions of high- and low mutation frequency which are lesion- and gene-specific [20]
To counter the mutagenic effect of 8-oxoG, Escherichia coli has evolved an elaborate repair system consisting of three genes, mutT, mutM, and mutY [7, 11]. mutT codes for a sanitizing enzyme that converts 8-oxoGTP in the nucleotide pool to 8-oxoGMP, preventing the incorporation of 8-oxoG into DNA during replication [12]. mutM or fpg codes for formamidopyrimidine DNA N-glycosylase (Fpg) [13], a protein with N-glycosylase and AP lyase activities [14, 15] that acts on both purine and pyrimidine oxidation products [16]
Fpg protein was overexpressed in E. coli and purified following the procedure described for endonuclease VIII [33]
Summary
Chemicals and Enzymes. 8-oxoG phosphoramidite was from Glen Research (Sterling, VA). Wild-type T4 gp, gp43 deficient in the 3Ј 3 5Ј proofreading exonuclease activity (gp exoϪ), T4 gp, gp62, gp, and gp were prepared as described [30,31,32]. The lesion-containing strand was 5Ј end-labeled with 32P, annealed to its complement, adjusted to a range of molar concentrations between 0.5 and 105 nM, and subjected to cleavage by Fpg (final concentration 0.25 nM) at 37°C in 10 mM Tris1⁄7HCl (pH 7.5), 1 mM EDTA, and 50 mM NaCl. Following product analysis by denaturing PAGE and quantification by molecular imaging, the reactions’ initial rates were determined and plotted as a function of substrate concentration. The 54 mers containing 8-oxoG at positions 1 and 2 and the 24 mer containing the lesion at position 4 (see above) were used as templates to determine the steady-state kinetics of nucleotide incorporation and extension opposite 8-oxoG by gp exoϪ. Single nucleotide incorporation rates were determined as a function of dNTP concentration, and the steady-state rate constants were derived as described for Fpg kinetics. The concentration of active molecules was likely lower than template concentration because the reaction’s initial velocity could be measured in a time scale of 5 to 60 s even at the highest dNTP concentration used [under polymerase excess conditions, DNA synthesis across 8-oxoG has been shown to proceed at a rate of 10s of nucleotides per second [36], making it impossible to measure an initial rate within the time scale used in our experiments]
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