Reduced sulfhydryls maintain specific incision of BPDE-DNA adducts by recombinant thermoresistant Bacillus caldotenax UvrABC endonuclease.
Prokaryotic DNA repair nucleases are useful reagents for detecting DNA lesions. Escherichia coli UvrABC endonuclease can incise DNA containing UV photoproducts and bulky chemical adducts. The limited stability of the E. coli UvrABC subunits leads to difficulty in estimating incision efficiency and quantitative adduct detection. To develop a more stable enzyme with greater utility for the detection of DNA adducts, thermoresistant UvrABC endonuclease was cloned from the eubacterium Bacillus caldotenax (Bca) and individual recombinant protein subunits were overexpressed in and purified from E. coli. Here, we show that Bca UvrC that had lost activity or specificity could be restored by dialysis against buffer containing 500 mM KCl and 20mM dithiothreitol. Our data indicate that UvrC solubility depended on high salt concentrations and UvrC nuclease activity and the specificity of incisions depended on the presence of reduced sulfhydryls. Optimal conditions for BCA UvrABC-specific cleavage of plasmid DNAs treated with [3H](+)-7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) (1-5 lesions/plasmid) were developed. Preincubation of substrates with UvrA and UvrB enhanced incision efficiency on damaged substrates and decreased non-specific nuclease activity on undamaged substrates. Under optimal conditions for damaged plasmid incision, approximately 70% of adducts were incised in 1 nM plasmid DNA (2 BPDE adducts/5.4 kbp plasmid) with UvrA at 2.5 nM, UvrB at 62.5 nM, and UvrC at 25 nM. These results demonstrate the potential usefulness of the Bca UvrABC for monitoring the distribution of chemical carcinogen-induced lesions in DNA.
Jiang
GuoHui
G
Department of Pharmacology and Toxicology, University of Louisville, 570 S. Preston St., Suite 221, Louisville, KY 40202, USA.
Skorvaga
Milan
M
Van Houten
Bennett
B
States
J Christopher
JC
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000032:analysis / Q000737:chemistry*; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D002686:Chitin / Q000737:chemistry; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D018736:DNA Adducts / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D004249:DNA Damage; D004265:DNA Helicases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D004260:DNA Repair; D004278:DNA, Superhelical / Q000737:chemistry; D003851:Deoxyribonucleases / Q000378:metabolism; D004229:Dithiothreitol / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004706:Endodeoxyribonucleases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics; D006358:Hot Temperature; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D009690:Nucleic Acid Conformation / Q000187:drug effects; D010957:Plasmids / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors
D018736:DNA Adducts; D004278:DNA, Superhelical; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; C058093:UvrB protein, E coli; D002686:Chitin; D007544:Isopropyl Thiogalactoside; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D003851:Deoxyribonucleases; D004706:Endodeoxyribonucleases; C032783:UV incision nuclease; C066567:UvrC protein, E coli; C036385:endodeoxyribonuclease uvrABC; D004265:DNA Helicases; D004229:Dithiothreitol