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1,N 6 -α-hydroxypropanoadenine, the acrolein adduct to adenine, is a substrate for AlkB dioxygenase

Dylewska, Małgorzata and Kusmierek, Jaroslaw T. and Pilżys, Tomasz and Poznański, Jarosław and Maciejewska, Agnieszka M. (2017) 1,N 6 -α-hydroxypropanoadenine, the acrolein adduct to adenine, is a substrate for AlkB dioxygenase. Biochemical Journal, 474 (11). pp. 1837-1852. ISSN 0264-6021

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Official URL: http://doi.org/10.1042/BCJ20161008


1,N6-α-hydroxypropanoadenine (HPA) is an exocyclic DNA adduct of acrolein – an environmental pollutant and endocellular oxidative stress product. Escherichia 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 determined. 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 stereoisomer 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 identification of the active protein form, with cofactor and cosubstrate bound, and monitoring of substrate binding. In contrast FTO, a human AlkB homolog, failed to bind an ssDNA trimer carrying HPA.

Item Type:Article
Uncontrolled Keywords:1,N6-α-hydroxypropanoadenine, AlkB dioxygenase, acrolein, mutagenesis, DNA repair, substrate binding
Subjects:Q Science > Q Science (General)
Divisions:Department of Biophysics
Department of Molecular Biology
ID Code:1363
Deposited By: Prof Jaroslaw Poznanski
Deposited On:24 Jul 2017 08:00
Last Modified:14 Apr 2018 22:05

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