Jiang GuoHui, Skorvaga Milan, Van Houten Bennett, States J Christopher
Department of Pharmacology and Toxicology, University of Louisville, 570 S. Preston St., Suite 221, Louisville, KY 40202, USA.
Protein Expr Purif. 2003 Sep;31(1):88-98. doi: 10.1016/s1046-5928(03)00137-2.
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.
原核生物DNA修复核酸酶是检测DNA损伤的有用试剂。大肠杆菌UvrABC核酸内切酶可切割含有紫外线光产物和大分子化学加合物的DNA。大肠杆菌UvrABC亚基稳定性有限，导致难以评估切割效率和进行加合物定量检测。为了开发一种更稳定且在检测DNA加合物方面更有用的酶，从嗜热芽孢杆菌（Bca）中克隆了耐热UvrABC核酸内切酶，并在大肠杆菌中过表达和纯化了各个重组蛋白亚基。在此，我们表明，失活或丧失特异性的Bca UvrC可通过在含有500 mM KCl和20 mM二硫苏糖醇的缓冲液中透析来恢复。我们的数据表明，UvrC的溶解度取决于高盐浓度，UvrC核酸酶活性和切割特异性取决于还原巯基的存在。确定了用3H-7R,8S-二羟基-9S,10R-环氧-7,8,9,10-四氢苯并[a]芘（BPDE）（1 - 5个损伤/质粒）处理的质粒DNA进行BCA UvrABC特异性切割的最佳条件。底物与UvrA和UvrB预孵育可提高对受损底物的切割效率，并降低对未受损底物的非特异性核酸酶活性。在受损质粒切割的最佳条件下，对于1 nM质粒DNA（2个BPDE加合物/5.4 kbp质粒），在2.5 nM的UvrA、62.5 nM的UvrB和25 nM的UvrC存在下，约70%的加合物被切割。这些结果证明了Bca UvrABC在监测化学致癌物诱导的DNA损伤分布方面的潜在用途。
