Escherichia coli nucleoside diphosphate kinase does not act as a uracil-processing DNA repair nuclease

doi:10.1073/pnas.0401031101

Escherichia coli nucleoside diphosphate kinase does not act as a uracil-processing DNA repair nuclease

Departments of ^*Environmental and Molecular Toxicology and ^§Biochemistry and Biophysics and ^†Environmental Health Science Center, Oregon State University, Corvallis, OR 97331-7301

Edited by Richard D. Kolodner, University of California at San Diego School of Medicine, La Jolla, CA (received for review February 13, 2004)

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.

Footnotes

↵ ‡ To whom correspondence should be addressed. E-mail: bennetsa{at}onid.oregonstate.edu.
↵ ¶ Deceased February 17, 2004.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: Ndk, Escherichia coli nucleoside diphosphate kinase; BER, base excision repair; Ung, E. coli uracil-DNA glycosylase; Dug, E. coli double-stranded uracil-DNA glycosylase; Ugi, uracil-DNA glycosylase inhibitor protein; AP, apurinic/apyrimidinic.