Abstract
AlkB is a bacterial Fe(II)- and 2-oxoglutarate-dependent dioxygenase that repairs a wide range of alkylated nucleobases in DNA and RNA as part of the adaptive response to exogenous nucleic acid-alkylating agents. Although there has been longstanding interest in the structure and specificity of Escherichia coli AlkB and its homologs, difficulties in assaying their repair activities have limited our understanding of their substrate specificities and kinetic mechanisms. Here, we used quantitative kinetic approaches to determine the transient kinetics of recognition and repair of alkylated DNA by AlkB. These experiments revealed that AlkB is a much faster alkylation repair enzyme than previously reported and that it is significantly faster than DNA repair glycosylases that recognize and excise some of the same base lesions. We observed that whereas 1,N6-ethenoadenine can be repaired by AlkB with similar efficiencies in both single- and double-stranded DNA, 1-methyladenine is preferentially repaired in single-stranded DNA. Our results lay the groundwork for future studies of AlkB and its human homologs ALKBH2 and ALKBH3.
Highlights
AlkB is a bacterial Fe(II)– and 2-oxoglutarate– dependent dioxygenase that repairs a wide range of alkylated nucleobases in DNA and RNA as part of the adaptive response to exogenous nucleic acid–alkylating agents
This consistency validates the use of the single turnover specificity constant kmax/K1⁄2 to evaluate the substrate specificity of AlkB, as an alternative to multiple-turnover measurements that are adversely affected by AlkB inactivation
A wide variety of alkylated nucleobases have been shown to be substrates of AlkB [2], but a quantitative understanding of its substrate specificity has been impeded by differences in how studies have been performed
Summary
Recombinant E. coli AlkB was purified without metal and stored in 1 mM EDTA to ensure protein stability. To evaluate the quality of the purified enzyme, we compared the total concentration of AlkB, as determined by its absorbance at 280 nm and its predicted extinction coefficient, with the total concentration of metal that it binds, using ICP-MS to perform quantitative metal analysis.
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