Reduced sulfhydryls maintain specific incision of BPDE–DNA adducts by recombinant thermoresistant Bacillus caldotenax UvrABC endonuclease
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- 作者：Jiang ; GuoHui ; Skorvaga ; Milan ; Van Houten ; Bennett ; States ; J. Christopher
- 关键词：DNA damage ; Benzo[a]pyrene-diol-epoxide ; DNA repair ; UvrABC endonuclease
- 刊名：Protein Expression and Purification
- 出版年：2003
- 出版时间：September, 2003
- 年：2003
- 卷：31
- 期：1
- 页码：88-98
- 全文大小：272 K
文摘
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 500mM 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, 
70 % of adducts were incised in 1nM plasmid DNA (2 BPDE adducts/5.4kbp plasmid) with UvrA at 2.5nM, UvrB at 62.5nM, and UvrC at 25nM. These results demonstrate the potential usefulness of the Bca UvrABC for monitoring the distribution of chemical carcinogen-induced lesions in DNA.
70 % of adducts were incised in 1nM plasmid DNA (2 BPDE adducts/5.4kbp plasmid) with UvrA at 2.5nM, UvrB at 62.5nM, and UvrC at 25nM. These results demonstrate the potential usefulness of the Bca UvrABC for monitoring the distribution of chemical carcinogen-induced lesions in DNA.