title: Escherichia coli nucleoside diphosphate kinase does not act as a uracil-processing DNA repair nuclease.
abstract: Escherichia coli nucleoside diphosphate kinase (Ndk) catalyzes ATP-dependent synthesis of ribo- and deoxyribonucleoside triphosphates from the cognate diphosphate precursor. Recently, the Ndk polypeptide was reported to be a multifunctional base excision repair nuclease that processed uracil residues in DNA by acting sequentially as a uracil-DNA glycosylase inhibitor protein (Ugi)-sensitive uracil-DNA glycosylase, an apurinic/apyrimidiniclyase, and a 3'-phosphodiesterase [Postel, E. H. & Abramczyk, B. M. (2003) Proc. Natl. Acad. Sci. USA 100, 13247-13252]. Here we demonstrate that the E. coli Ndk polypeptide lacked detectable uracil-DNA glycosylase activity and, hence, was incapable of acting as a uracil-processing DNA repair nuclease. This finding was based on the following observations: (i) uracil-DNA glycosylase activity did not copurify with Ndk activity; (ii) Ndk purified from E. coli ung(-) cells showed no detectable uracil-DNA glycosylase activity; and (iii) Ndk failed to bind to a Ugi-Sepharose affinity column that tightly bound E. coli uracil-DNA glycosylase (Ung). Collectively, these observations demonstrate that the E. coli Ndk polypeptide does not possess inherent uracil-DNA glycosylase activity.
authors.affiliation: Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR 97331-7301, USA. bennetsa@onid.oregonstate.edu
authors.forename: Samuel E
authors.initials: SE
authors.lastname: Bennett
authors.forename: Cheng-Yao
authors.initials: CY
authors.lastname: Chen
authors.forename: Dale W
authors.initials: DW
authors.lastname: Mosbaugh
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D017931:DNA Primers; D004260:DNA Repair; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D009701:Nucleoside-Diphosphate Kinase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D017931:DNA Primers; D009701:Nucleoside-Diphosphate Kinase