Robust incision of Benoz[a]pyrene-7,8-dihyrodiol-9,10-epoxide-DNA adducts by a recombinant thermoresistant interspecies combination UvrABC endonuclease system.
Prokaryotic DNA repair nucleases are useful reagents for detecting DNA lesions. UvrABC endonuclease, encoded by the UvrA, UvrB, and UvrC genes can incise DNA containing bulky nucleotide adducts and intrastrand cross-links. UvrA, UvrB, and UvrC were cloned from Bacillus caldotenax (Bca)and UvrC from Thermatoga maritima (Tma), and recombinant proteins were overexpressed in and purified from Escherichia coli. Incision activities of UvrABC composed of all Bca-derived subunits (UvrABC(Bca)) and an interspecies combination UvrABC composed of Bca-derived UvrA and UvrB and Tma-derived UvrC (UvrABC(Tma)) were compared on benoz[a]pyrene-7,8-dihyrodiol-9,10-epoxide (BPDE)-adducted substrates. Both UvrABC(Bca) and UvrABC(Tma) specifically incised both BPDE-adducted plasmid DNAs and site-specifically modified 50-bp oligonucleotides containing a single (+)-trans- or (+)-cis-BPDE adduct. Incision activity was maximal at 55-60 degrees C. However, UvrABC(Tma) was more robust than UvrABC(Bca) with 4-fold greater incision activity on BPDE-adducted oligonucleotides and 1.5-fold greater on [(3)H]BPDE-adducted plasmid DNAs. Remarkably, UvrABC(Bca) incised only at the eighth phosphodiester bond 5' to the BPDE-modified guanosine. In contrast, UvrABC(Tma) performed dual incision, cutting at both the fifth phosphodiester bond 3' and eighth phosphodiester bond 5' from BPDE-modified guanosine. BPDE adduct stereochemistry influenced incision activity, and cis adducts on oligonucleotide substrates were incised more efficiently than trans adducts by both UvrABC(Bca) and UvrABC(Tma). UvrAB-DNA complex formation was similar with (+)-trans- and (+)-cis-BPDE-adducted substrates, suggesting that UvrAB binds both adducts equally and that adduct configuration modifies UvrC recognition of the UvrAB-DNA complex. The dual incision capabilities and higher incision activity of UvrABC(Tma) make it a robust tool for DNA adduct studies.
Jiang
Guo Hui
GH
Department of Pharmacology and Toxicology, Brown Cancer Center, and Center for Genetics and Molecular Medicine, University of Louisville, Louisville, Kentucky 40202, USA.
Skorvaga
Milan
M
Croteau
Deborah L
DL
Van Houten
Bennett
B
States
J Christopher
JC
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D018736:DNA Adducts / Q000737:chemistry / Q000378:metabolism*; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair / Q000502:physiology*; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D020124:Thermotoga maritima / Q000201:enzymology*
D018736:DNA Adducts; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C058093:UvrB protein, E coli; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D004706:Endodeoxyribonucleases; C066567:UvrC protein, E coli; C036385:endodeoxyribonuclease uvrABC; D004265:DNA Helicases