1,N6-α-hydroxypropanoadenine (HPA) is an exocyclic DNA adduct of acrolein - an environmental pollutant and endocellular oxidative stress product. E. coli AlkB dioxygenase belongs to the superfamily of α-ketoglutarate (αKG)- and iron-dependent dioxygenases which remove alkyl lesions from bases via an oxidative mechanism, thereby restoring native DNA structure. Here, we provide in vivo and in vitro evidence that HPA is mutagenic and is effectively repaired by AlkB dioxygenase. HPA generated in plasmid DNA caused A→C and A→T transversions and, less frequently, A→G transitions. The lesion was efficiently repaired by purified AlkB protein; the optimal pH, Fe(II) and αKG concentrations for this reaction were determinated. In vitro kinetic data show that the protonated form of HPA is preferentially repaired by AlkB, albeit the reaction is stereoselective. Moreover, the number of reaction cycles carried out by an AlkB molecule remains limited. Molecular modeling of the T(HPA)T/AlkB complex demonstrated that the R stereoisomer in the equatorial conformation of the HPA hydroxyl group is strongly preferred, while the S one seems to be susceptible to AlkB-directed oxidative hydroxylation only when HPA adopts the syn conformation around the glycosidic bond. In addition to the biochemical activity assays, substrate binding to the protein was monitored by differential scanning fluorimetry allowing the identification of the active protein form with cofactor and cosubstrate bound and monitoring substrate binding. In contrast FTO, a human AlkB homolog, failed bind an ssDNA trimer carrying HPA.
- AlkB dioxygenase
- DNA repair
- substrate binding
- ©2017 The Author(s)
This is an Accepted Manuscript; not the final Version of Record. Archiving permitted only in line with the archiving policy of Portland Press Limited. All other rights reserved.