Escherichia coli double-strand uracil-DNA glycosylase: involvement in uracil-mediated DNA base excision repair and stimulation of activity by endonuclease IV.
Escherichia coli double-strand uracil-DNA glycosylase (Dug) was purified to apparent homogeneity as both a native and recombinant protein. The molecular weight of recombinant Dug was 18 670, as determined by matrix-assisted laser desorption-ionization mass spectrometry. Dug was active on duplex oligonucleotides (34-mers) that contained site-specific U.G, U.A, ethenoC.G, and ethenoC.A targets; however, activity was not detected on DNA containing a T.G mispair or single-stranded DNA containing either a site-specific uracil or ethenoC residue. One of the distinctive characteristics of Dug was that the purified enzyme excised a near stoichiometric amount of uracil from U.G-containing oligonucleotide substrate. Electrophoretic mobility shift assays revealed that the lack of turnover was the result of strong binding by Dug to the reaction product apyrimidinic-site (AP) DNA. Addition of E. coli endonuclease IV stimulated Dug activity by enhancing the rate and extent of uracil excision by promoting dissociation of Dug from the AP. G-containing 34-mer. Catalytically active endonuclease IV was apparently required to mediate Dug turnover, since the addition of 5 mM EDTA mitigated the effect. Further support for this interpretation came from the observations that Dug preferentially bound 34-mer containing an AP.G target, while binding was not observed on a substrate incised 5' to the AP-site. We also investigated whether Dug could initiate a uracil-mediated base excision repair pathway in E. coli NR8052 cell extracts using M13mp2op14 DNA (form I) containing a site-specific U.G mispair. Analysis of reaction products revealed a time dependent appearance of repaired form I DNA; addition of purified Dug to the cell extract stimulated the rate of repair.
Sung
J S
JS
Departments of Environmental and Molecular Toxicology and Biochemistry and Biophysics and the Environmental Health Science Center, Oregon State University, Corvallis 97731, USA.
Mosbaugh
D W
DW
D020137:Base Pair Mismatch; D019757:Carbon-Oxygen Lyases / Q000378:metabolism*; D003001:Cloning, Molecular; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D008970:Molecular Weight; D009699:N-Glycosyl Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; D009699:N-Glycosyl Hydrolases; C412878:double-strand uracil-DNA glycosylase, E coli; D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase