DNA glycosylases that remove alkylated and deaminated purine nucleobases are essential DNA repair enzymes that protect the genome, and at the same time confound cancer alkylation therapy, by excising cytotoxic N3-methyladenine bases formed by DNA targeting anticancer compounds. The basis for glycosylase specificity toward N3- and N7-alkylpurines is believed to result from intrinsic instability of the modified bases and not from direct enzyme functional group chemistry. Here, we present crystal structures of the recently discovered Bacillus cereus AlkD glycosylase in complex with DNAs containing alkylated, mismatched, and abasic nucleotides. Unlike other glycosylases, AlkD captures the extrahelical lesion in a solvent-exposed orientation, providing the first illustration for how hydrolysis of N3- and N7-alkylated bases may be facilitated by increased lifetime out of the DNA helix. The structures and supporting biochemical analysis of base flipping and catalysis reveal how AlkD’s HEAT-repeats distort the DNA backbone to detect non-Watson-Crick base pairs without duplex intercalation.

Hepatitis B virus (HBV) replicates its DNA genome through reverse transcription of a pregenomic RNA (pgRNA) by using a multifunctional polymerase (HP). A critical function of HP is its specific recognition of a viral RNA signal termed ε (Hε) located on pgRNA, which is required for specific packaging of pgRNA into viral nucleocapsids and initiation of viral reverse transcription. HP initiates reverse transcription by using itself as a protein primer (protein priming) and Hε as the obligatory template. We have purified HP from human cells that retained Hε binding activity in vitro . Furthermore, HP purified as a complex with Hε, but not HP alone, displayed in vitro protein priming activity. While the HP-Hε interaction in vitro and in vivo required the Hε internal bulge, but not its apical loop, and was not significantly affected by the cap-Hε distance, protein priming required both the Hε apical loop and internal bulge, as well as a short distance between the cap and Hε, mirroring the requirements for RNA packaging. These studies have thus established new HBV protein priming and RNA binding assays that should greatly facilitate the dissection of the requirements and molecular mechanisms of HP-Hε interactions, RNA packaging, and protein priming.

Sirtuin 3 (Sirt3), a major mitochondrial NAD+-dependent deacetylase, targets various mitochondrial proteins for lysine deacetylation and regulates important cellular functions such as energy metabolism, aging, and stress response. In this study, we identified the human 8-oxoguanine-DNA glycosylase 1 (OGG1), a DNA repair enzyme that excises 7,8-dihydro-8-oxoguanine (8-oxoG) from damaged genome, as a new target protein for Sirt3. We found that Sirt3 physically associated with OGG1 and deacetylated this DNA glycosylase and that deacetylation by Sirt3 prevented the degradation of the OGG1 protein and controlled its incision activity. We further showed that regulation of the acetylation and turnover of OGG1 by Sirt3 played a critical role in repairing mitochondrial DNA (mtDNA) damage, protecting mitochondrial integrity, and preventing apoptotic cell death under oxidative stress. We observed that following ionizing radiation, human tumor cells with silencing of Sirt3 expression exhibited deteriorated oxidative damage of mtDNA, as measured by the accumulation of 8-oxoG and 4977 common deletion, and showed more severe mitochondrial dysfunction and underwent greater apoptosis in comparison with the cells without silencing of Sirt3 expression. The results reported here not only reveal a new function and mechanism for Sirt3 in defending the mitochondrial genome against oxidative damage and protecting from the genotoxic stress-induced apoptotic cell death but also provide evidence supporting a new mtDNA repair pathway.

The lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is activated to reactive metabolites that methylate or pyridyloxobutylate DNA. Previous studies demonstrated that pyridyloxobutylated DNA interferes with the repair of O6-methylguanine (O6-mG) by O6-alkylguanine-DNA alkyltransferase (AGT). The AGT reactivity of pyridyloxobutylated DNA was attributed to (pyridyloxobutyl)guanine adducts. One potential AGT substrate adduct, 2'-deoxy-O6-[4-oxo-4-(3-pyridyl)butyl]guanosine (O6-pobdG), was prepared. This adduct was stable at pH 7.0 for greater than 13 days and to neutral thermal hydrolysis conditions (pH 7.0, 100 degrees C, 30 min). Under mild acid hydrolysis conditions (0.1 N HCl, 80 degrees C), O6-pobdG was depurinated to yield O6-[4-oxo-4-(3-pyridyl)butyl]guanine (O6-pobG). O6-pobdG was hydrolyzed to 4-hydroxy-1-(3-pyridyl)-1-butanone and guanine under strong acid hydrolysis conditions (0.8 N HCl, 80 degrees C). O6-pobG was detected in 0.1 N HCl hydrolysates of DNA alkylated with the model pyridyloxobutylating agent 4-(acetoxymethylnitrosamino)-1-(3-[5-3H]pyridyl)-1-butanone ([5-3H]NNKOAc). When [5-3H]NNKOAc-treated DNA was incubated with either rat liver or recombinant human AGT, O6-pobG was removed, presumably a result of transfer of the pyridyloxobutyl group from the O6-position of guanine to AGT's active site.

DNA was isolated from tissues of F344 rats 24 h after treatment by s.c. injection with [5-3H]4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone ([5-3H]NNK) or [5-3H]N'-nitrosonornicotine ([5-3H]NNN). It was hydrolyzed with acid or at pH 7, 100 degrees C, and the hydrolysates were analyzed by HPLC. The major product in each case was identified as 4-hydroxy-1-(3-pyridyl)-1-butanone, formed by hydrolysis of a DNA adduct. It was detected in DNA from the livers of rats treated with [5-3H]NNK or [5-3H]NNN, and in DNA from lungs of rats treated with [5-3H]NNK. These results demonstrate that 4-(3-pyridyl)-4-oxobutylation of DNA occurs in rats treated with NNK or NNN, and are consistent with the hypothesis that these nitrosamines are metabolically activated by alpha-hydroxylation.

DNA polymerases accurately replicate DNA by incorporating mostly correct dNTPs opposite any given template base. We have identified the chemical features of purine dNTPs that human pol α uses to discriminate between right and wrong dNTPs. Removing N-3 from guanine and adenine, two high fidelity bases, significantly lowers fidelity. Analogously, adding the equivalent of N-3 to lowfidelity benzimidazole-derived bases (i.e., bases that pol α rapidly incorporates opposite all 4 natural bases) and to generate 1-deazapurines significantly increases the ability of pol α to identify the resulting 1-deazapurines as wrong. Adding the equivalent of the purine N-1 to benzimidazole or to 1-deazapurines significantly decreases the rate at which pol α polymerizes the resulting bases opposite A, C, and G, while simultaneously enhancing polymerization opposite T. Conversely, adding the equivalent of adenine's C-6 exocyclic amine (N-6) to 1-and 3-deazapurines also enhances polymerization opposite T, but does not significantly decrease polymerization opposite A, C, and G. Importantly, if the newly inserted bases lack N-1 and N-6, pol α does not efficiently polymerize the next correct dNTP, whereas if it lacks N-3 one additional nucleotide is added and then chain termination ensues. These data indicate that pol α uses two orthogonal screens to maximize its fidelity. During dNTP polymerization, it uses a combination of negative (N-1 and N-3) and positive (N-1 and † This work was supported by grants to RDK from the NIH (GM54194 and TW007372-01) and the Army Research Office (W911NF-05-1-0172), to MH from the Ministry of Education of the Czech Republic (Centre of Biomolecules and Complex Molecular Systems, LC 512), and to JE from the Deutsche Forschungsgemeinschaft (SFB 579).

DNA polymerases replicate DNA with high fidelity despite the small differences in energy between correct and incorrect base pairs. X-ray crystallographic and structure-activity kinetic experiments have implicated interactions with the minor groove of the DNA as being crucial for catalysis and fidelity. The current hypothesis is that polymerases check the geometry of the base pairs through hydrogen bonds and steric interactions with the minor groove of the DNA. The mechanisms by which these interactions are related to catalysis and fidelity are not known. In this manuscript, we have studied these interactions using a combination of site-specific mutagenesis of Escherichia coli DNA polymerase I (Klenow fragment) and atomic substitution of the DNA. Crystal structures have predicted hydrogen bonds from Arg668 to the terminal base on the primer (P1) and Gln849 to its base pair partner (T1). Kinetic studies, however, have implicated the minor groove of the primer terminus but not its base pair partner as being important to catalysis and fidelity. Hydrogen bonds between Arg668 and Gln849 to the DNA were probed with the site specific mutants, R668A and Q849A. Hydrogen bonds from the DNA were probed with three oligodeoxynucleotides which have a guanine or 3-deazaguanine (3DG) at P1, T1, or T2. We found that the pre-steady-state parameter k(pol) was decreased with R668A (40-fold) and Q849A (150-fold) or with 3DG at P1 (300-fold) or T2 (25-fold). When R668A was combined with 3DG at P1 the decrease in rate was only 80-fold, consistent with a hydrogen bond between Arg668 and P1. In contrast, when the 3DG substitution at P1 was combined with Q849A the rate reduction was 15000-fold. Similar reactions between R668A or Q849A and T2 showed that there are interactions between these sites although the interactions are not as strong as between P1 and R668.

O6-alkylguanine-DNA alkyltransferase (AGT) repairs O6-alkylguanine residues at different rates depending on the identity of the alkyl group as well as the sequence context. To elucidate the mechanism(s) underlying the differences in rates, we examined the repair of five alkyl groups in three different sequence contexts. The kinact and Km values were determined by measuring the rates of repair of oligodeoxynucleotide duplexes containing the O6-alkylguanine residues with various concentrations of AGT in excess. The time course of the reactions all followed pseudo-first-order kinetics except for one of the O6-ethylguanine substrates, which could be analyzed in a two-phase exponential equation. The differences in rates of repair between the different alkyl groups and the different sequence contexts are dependent on rates of alkyl transfer and not substrate recognition. The relative rates of reaction are in general benzyl>methyl>ethyl>2-hydroxyethyl>4-(3-pyridyl)-4-oxobutyl, but the absolute rates are dependent on sequence. The kinact values between benzyl and 4-(3-pyridyl)-4-oxobutyl range from 2300 to 350000 depending on sequence. The sequence-dependent variation in kinact varied the most for O6-[4-(3-pyridyl)-4-oxobutyl]guanine, which ranged from 0.022 to 0.000016 s(-1). The results are consistent with a mechanism in which the O6-alkylguanine can bind to AGT in either a reactive or an unreactive orientation, the proportion of which depends on the sequence context.