title: Characterization of a Trypanosoma brucei Alkb homolog capable of repairing alkylated DNA.
abstract: Trypanosoma brucei encodes a protein (denoted TbABH) that is homologous to AlkB of Escherichia coli and AlkB homolog (ABH) proteins in other organisms, raising the possibility that trypanosomes catalyze oxidative repair of alkylation-damaged DNA. TbABH was cloned and expressed in E. coli, and the recombinant protein was purified and characterized. Incubation of anaerobic TbABH with Fe(II) and α-ketoglutarate (αKG) produces a characteristic metal-to-ligand charge-transfer chromophore, confirming its membership in the Fe(II)/αKG dioxygenase superfamily. The protein binds to DNA, with a clear preference for alkylated oligonucleotides according to results derived by electrophoretic mobility shift assays. Finally, the protozoan gene was shown to partially complement E. coli alkB cells when stressed with methylmethanesulfonate; thus confirming assignment of TbABH as a functional AlkB protein in T. brucei.
authors.affiliation: Department of Biochemistry & Molecular Biology, Michigan State University, East Lansing, MI, United States.
authors.forename: Jana M
authors.initials: JM
authors.lastname: Simmons
authors.forename: Donna J
authors.initials: DJ
authors.lastname: Koslowsky
authors.forename: Robert P
authors.initials: RP
authors.lastname: Hausinger
mesh_terms: D000478:Alkylation; D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004260:DNA Repair / Q000502:physiology*; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli; D015870:Gene Expression; D005816:Genetic Complementation Test; D006801:Humans; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D013056:Spectrophotometry, Ultraviolet; D014346:Trypanosoma brucei brucei / Q000737:chemistry / Q000235:genetics*
chemical_list: D016054:DNA, Protozoan; D004268:DNA-Binding Proteins; D015800:Protozoan Proteins; D011994:Recombinant Proteins