Escherichia coli nucleoside diphosphate kinase is a uracil-processing DNA repair nuclease

Abstract

Escherichia coli nucleoside diphosphate kinase (eNDK) is an XTP:XDP phosphotransferase that plays an important role in the regulation of cellular nucleoside triphosphate concentrations. It is also one of several recently discovered DNases belonging to the NM23/NDK family. E. coli cells disrupted in the ndk gene display a spontaneous mutator phenotype, which has been attributed to the mutagenic effects of imbalanced nucleotide pools and errors made by replicative DNA polymerases. Another explanation for the increased mutation rates is that endk- cells lack the nuclease activity of the NDK protein that is essential for a DNA repair pathway. Here, we show that purified, cloned endk is a DNA repair nuclease whose substrate is uracil misincorporated into DNA. We have identified three new catalytic activities in eNDK that act sequentially to repair the uracil lesion: (i) uracil-DNA glycosylase that excises uracil from single-stranded and from U/A and U/G mispairs in double-stranded DNA; (ii) apyrimidinic endonuclease that cleaves double-stranded DNA as a lyase by forming a covalent enzyme-DNA intermediate complex with the apyrimidinic site created by the glycosylase; and (iii) DNA repair phosphodiesterase that removes 3'-blocking residues from the ends of duplex DNA. All three of these activities, as well as the nucleoside-diphosphate kinase, reside in the same protein. Based on these findings, we propose an editing function for eNDK as a mechanism by which the enzyme prevents mutations in DNA.

Fig. 1.

Uracil processing by eNDK. (A) Schematic view of the oligonucleotide substrate used in this experiment. Asterisk indicates the radioactively labeled terminus. (B) DNA cleavage analysis of U/A, T/A, ssU (single-stranded), and U/G oligonucleotides. Sequencing ladders prepared from the T/A substrate (partial sequence shown here) were run alongside samples. Enzymes and substrates used are indicated above lanes. The smeared appearance of UNG-treated DNA is because of instability of AP DNA and to the collapse of the helix from loss of base-stacking interactions. Reaction mixtures were separated on 20% sequencing gels, and the gels were subjected to autoradiography. (C) DNA cleavage analysis using the UDG inhibitor protein Ugi. See text for details.

Fig. 2.

UDG activity of eNDK. Oligonucleotide-cleavage assay was carried out with the U/A oligonucleotide substrate in the presence of EDTA to prevent cleavage of the backbone by eNDK. Enzymes and substrates used are indicated above lanes. Reaction mixtures were separated on 20% sequencing gels, and the gels were subjected to autoradiography. See text for further details.

Fig. 3.

AP endonuclease activity of eNDK. (A) Schematic view of the oligonucleotide substrate used in this experiment, with the complementary strand having A opposite the AP site. Asterisk indicates the radioactively labeled terminus. (B) DNA cleavage analysis of AP-site DNA. Enzymes and substrates used are indicated above lanes. The smeared appearance of AP DNA by itself is because of its instability caused by the collapse of the helix (C). eNDK binds AP DNA covalently. Enzymes and substrates used are indicated above lanes. After reactions were stopped, samples were boiled for 10 min and then separated on SDS/15% PAGE gels followed by Coomassie blue staining and autoradiography. (D) Same as C, except only the AP substrate was used, and a duplicate gel was transferred to a PDF membrane without staining; after development, the membrane was also autoradiographed. Control lanes with eNDK alone in the absence of DNA did not produce radioactive signals (data not shown). See text for further details.

Fig. 4.

eNDK has 3′ repair phosphodiesterase activity. (A) Schematic view of oligonucleotide substrate used in this experiment. Asterisk indicates the radioactively labeled terminus. (B) Time course of phosphoglycolate removal from the 3′ end of the oligonucleotide by eNDK and EndoIV (0-20 min) and ExoIII (0-5 min). Enzymes and substrates used are indicated above lanes. Products are displayed on a 20% urea/PAGE gel. See text for further details.

Fig. 5.

Schematic of the proposed steps in eNDK-mediated uracil repair. Each step is detailed in the text.

Fig. 6.

Uracil processing (A), eNDK protein (B), and NDK activity (C) coelute from a DEAE column. Numbers shown under each panel represent protein-containing fractions. L, DEAE load; P, purified eNDK product. Substrate oligonucleotide used in the cleavage assay (A) was U/A.