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 ( . 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.

Molecular and Functional Interactions between Escherichia coli Nucleoside-diphosphate Kinase and the Uracil-DNA Glycosylase Ung

“…sensitive to the Ung-specific inhibitor Ugi (28). These findings were questioned on the basis that Ndk purified from Ungdeficient bacteria did not possess UDG activity (32,33). Our results here obtained from experiments using Ndks purified from various UDG-deficient mutant E. coli (ung Ϫ , ung Ϫ mug Ϫ , and ung ⌬ ) hosts and from the isogenic wild type confirm that the majority of UDG activity associated with Ndk is not an intrinsic property of the protein (Fig.…”

“…Oligonucleotide Substrate-5Ј-CCTGCCCTGUGCAGCT-GTGGG-3Ј (21-mer top strand) was radiolabeled using [␥- 32 P]ATP and T4 polynucleotide kinase. Double-stranded oligonucleotides were prepared by annealing the labeled top strand with a 1.5-fold molar excess of unlabeled complementary strand by heating for 10 min at 85°C and slow cooling.…”

“…One peak, representing ϳ30% of the total UDG activity, co-elutes with recombinant Ndk in the flow-through of this column, whereas the second larger peak, comprising ϳ70% of the activity, binds to the hydroxyapatite column from which it elutes with 40ϳ80 mM potassium phosphate. Under ordinary circumstances, Ndk and Ung should have been completely resolved by hydroxyapatite chromatography, as Ndk does not bind to hydroxyapatite at neutral pH (28,32), whereas Ung does (Ref. 32 and these results).…”

“…Prior to that, all of the uracil-DNA glycosylase (UDG) activity in extracts of E. coli has been attributed to either Ung, the major uracil-DNA glycosylase, or to Mug, an auxiliary enzyme (30,31). Furthermore, any association, intrinsic or extrinsic, of a uracil-DNA glycosylase activity with Ndk was ruled out by Bennett et al (32) and Kumar et al (33) on the bases that Ndk purified from Ungdeficient mutant bacteria did not possess UDG activity and that there was no evidence of a physical interaction between Ndk and Ung (32,33). Our attempts to resolve these controversial findings led to the observations reported in this paper, whereby the great majority of the uracil excision activity associated with purified Ndk is not an inherent property but rather is a direct consequence of the physical and functional interaction between Ung and Ndk.…”

Arabidopsis Uracil DNA Glycosylase (UNG) Is Required for Base Excision Repair of Uracil and Increases Plant Sensitivity to 5-Fluorouracil

“…Expression of recombinant AtUNG was carried out in E. coli BL21 (DE3) ung-151 cells (18). A fresh single transformant colony was inoculated into 10 ml of LB medium containing ampicillin (100 g/ml), tetracycline (10 g/ml), and chloramphenicol (34 g/ml), and the culture was incubated at 37°C overnight with shaking.…”