Human base excision repair creates a bias toward -1 frameshift mutations.

Frameshift mutations are particularly deleterious to protein function and play a prominent role in carcinogenesis. Most commonly these mutations involve the insertion or omission of a single nucleotide by a DNA polymerase that slips on a damaged or undamaged template. The mismatch DNA repair pathway can repair these nascent polymerase errors. However, overexpression of enzymes of the base excision repair (BER) pathway is known to increase the frequency of frameshift mutations suggesting competition between these pathways. We have examined the fate of DNA containing single nucleotide bulges in human cell extracts and discovered that several deaminated or alkylated nucleotides are efficiently removed by BER. Because single nucleotide bulges are more highly exposed we anticipate that they would be highly susceptible to spontaneous DNA damage. As a model for this, we have shown that chloroacetaldehyde reacts more than 18-fold faster with an A-bulge than with a stable A.T base pair to create alkylated DNA adducts that can be removed by alkyladenine DNA glycosylase. Reconstitution of the BER pathway using purified components establishes that bulged DNA is efficiently processed. Single nucleotide deletion is predicted to repair +1 frameshift events, but to make -1 frameshift events permanent. Therefore, these findings suggest an additional factor contributing to the bias toward deletion mutations.

Efficient recognition of an unpaired lesion by a DNA repair glycosylase.

The duplex structure of DNA, with its internal base pairing, protects the nucleobases from chemical damage, but it also poses a barrier to DNA-modifying enzymes, including the enzymes that recognize and repair DNA damage. It is known that unpaired (or bulged) nucleotides are significantly more accessible, but it is not known whether they might be recognized by nucleotide-flipping enzymes. We have investigated this question with human alkyladenine DNA glycosylase (AAG). AAG recognizes a wide variety of structurally disparate lesions, including deoxyinosine (I), which results from the spontaneous oxidative deamination of adenosine, and catalyzes the hydrolysis of the N-glycosidic bond to release the lesion base and initiate the base excision repair pathway. We used single-turnover kinetics to characterize the reactions of AAG with synthetic 25-mer oligonucleotides containing a single I lesion in single-stranded, mismatched, or single-nucleotide bulge contexts. We found that AAG has the highest catalytic efficiency toward a lesion that is presented in a single-nucleotide bulge. In contrast, AAG has more than 2000-fold reduced catalytic efficiency toward a single-stranded I-containing oligonucleotide relative to the duplexes. We have observed 20-fold differences in catalytic efficiency for the excision of the presumed biological target (paired with T) relative to alternative pairings such as C that might be formed by the replication of an unrepaired I. Furthermore, a linear free-energy relationship shows a strong inverse correlation between duplex stability and catalytic efficiency (slope = -0.6 to -1.0), indicating that gaining access to the base lesion provides a substantial barrier to AAG-catalyzed initiation of DNA repair. The observation that AAG recognizes a single-nucleotide bulge as efficiently as a mismatch implies that the recognition of DNA damage is remarkably plastic.

Characterization of alkyl-nickel adducts generated by reaction of methyl-coenzyme m reductase with brominated acids.

Methyl-coenzyme M reductase (MCR) from methanogenic archaea catalyzes the final step in the biological synthesis of methane. Using coenzyme B (CoBSH) as the two-electron donor, MCR reduces methyl-coenzyme M (methyl-SCoM) to methane and the mixed disulfide, CoB-S-S-CoM. MCR contains coenzyme F430, an essential redox-active nickel tetrahydrocorphin, at its active site. The active form of MCR (MCRred1) contains Ni(I)-F430. When 3-bromopropane sulfonate (BPS) is incubated with MCRred1, an alkyl-Ni(III) species is formed that elicits the MCRPS EPR signal. Here we used EPR and UV-visible spectroscopy and transient kinetics to study the reaction between MCR from Methanothermobacter marburgensis and a series of brominated carboxylic acids, with carbon chain lengths of 4-16. All of these compounds give rise to an alkyl-Ni intermediate with an EPR signal similar to that of the MCRPS species. Reaction of the alkyl-Ni(III) adduct, formed from brominated acids with eight or fewer total carbons, with HSCoM as nucleophile at pH 10.0 results in the formation of a thioether coupled to regeneration of the active MCRred1 state. When reacted with 4-bromobutyrate, MCRred1 forms the alkyl-Ni(III) MCRXA state and then, surprisingly, undergoes "self-reactivation" to regenerate the Ni(I) MCRred1 state and a bromocarboxy ester. The results demonstrate an unexpected reactivity and flexibility of the MCR active site in accommodating a broad range of substrates, which act as molecular rulers for the substrate channel in MCR.