Robust Incision of Benoz[a]pyrene-7,8-dihyrodiol-9,10-epoxide-DNA Adducts by a Recombinant Thermoresistant Interspecies Combination UvrABC Endonuclease System
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- 作者：Guo Hui Jiang ; Milan Skorvaga ; Deborah L. Croteau ; Bennett Van Houten ; and J. Christopher States
- 刊名：Biochemistry
- 出版年：2006
- 出版时间：June 27, 2006
- 年：2006
- 卷：45
- 期：25
- 页码：7834 - 7843
- 全文大小：450K
- 年卷期：v.45,no.25(June 27, 2006)
- ISSN：1520-4995
文摘
Prokaryotic DNA repair nucleases are useful reagents for detecting DNA lesions. UvrABCendonuclease, encoded by the UvrA, UvrB, and UvrC genes can incise DNA containing bulky nucleotideadducts 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 purifiedfrom Escherichia coli. Incision activities of UvrABC composed of all Bca-derived subunits (UvrABCBca)and an interspecies combination UvrABC composed of Bca-derived UvrA and UvrB and Tma-derivedUvrC (UvrABCTma) were compared on benoz[a]pyrene-7,8-dihyrodiol-9,10-epoxide (BPDE)-adductedsubstrates. Both UvrABCBca and UvrABCTma specifically incised both BPDE-adducted plasmid DNAsand site-specifically modified 50-bp oligonucleotides containing a single (+)-trans- or (+)-cis-BPDEadduct. Incision activity was maximal at 55-60 
C. However, UvrABCTma was more robust than UvrABCBcawith 4-fold greater incision activity on BPDE-adducted oligonucleotides and 1.5-fold greater on [3H]BPDE-adducted plasmid DNAs. Remarkably, UvrABCBca incised only at the eighth phosphodiester bond5' to the BPDE-modified guanosine. In contrast, UvrABCTma performed dual incision, cutting at both thefifth phosphodiester bond 3' and eighth phosphodiester bond 5' from BPDE-modified guanosine. BPDEadduct stereochemistry influenced incision activity, and cis adducts on oligonucleotide substrates wereincised more efficiently than trans adducts by both UvrABCBca and UvrABCTma. UvrAB-DNA complexformation was similar with (+)-trans- and (+)-cis-BPDE-adducted substrates, suggesting that UvrABbinds both adducts equally and that adduct configuration modifies UvrC recognition of the UvrAB-DNA complex. The dual incision capabilities and higher incision activity of UvrABCTma make it a robusttool for DNA adduct studies.
C. However, UvrABCTma was more robust than UvrABCBcawith 4-fold greater incision activity on BPDE-adducted oligonucleotides and 1.5-fold greater on [3H]BPDE-adducted plasmid DNAs. Remarkably, UvrABCBca incised only at the eighth phosphodiester bond5' to the BPDE-modified guanosine. In contrast, UvrABCTma performed dual incision, cutting at both thefifth phosphodiester bond 3' and eighth phosphodiester bond 5' from BPDE-modified guanosine. BPDEadduct stereochemistry influenced incision activity, and cis adducts on oligonucleotide substrates wereincised more efficiently than trans adducts by both UvrABCBca and UvrABCTma. UvrAB-DNA complexformation was similar with (+)-trans- and (+)-cis-BPDE-adducted substrates, suggesting that UvrABbinds both adducts equally and that adduct configuration modifies UvrC recognition of the UvrAB-DNA complex. The dual incision capabilities and higher incision activity of UvrABCTma make it a robusttool for DNA adduct studies.