Interaction of the UvrABC nuclease system with a DNA duplex containing a single stereoisomer of dG-(+)- or dG-(-)-anti-BPDE
- Univ. of Texas Medical Branch, Galveston, TX (United States)
Oligonucleotides containing site-specifically-modified N{sup 2}-guanine (+)-trans-, (-)-trans-, (+)-cis-, and (-)-cis-BPDE adducts into 50-base-pair DNA fragments. These substrates were used in reactions with the Escherichia coli UvrABC nuclease system. The interaction of the UvrA{sub 2} and UvrA{sub 2}B complexes with these four stereoisomers was probed using DNase I footprinting and gel mobility shift assays. DNase I digestion of substrates containing each stereoisomer of BPDE displayed a unique pattern which was consistent with the known structure of these DNA adducts. UvrA and UvrA{sub 2}B appeared to interact very similarly with all four substrates. Binding of UvrA{sub 2} to these substrates produced a 33-bp footprint, and the UvrB-DNA complex resulted in footprint of 24 bp. The UvrABC nuclease system produced bimodal incisions at the eighth phosphate 5{prime} and the fifth, sixth, or seventh phosphate 3{prime} to the modified guanine. The variation of the 3{prime} incision site was linked to the stereochemistry and orientation of the BPDE adduct. For example, the 3{prime} incision of the 50-bp duplex containing (-)-trans-BPDE-N{sup 2}-guanine was inhibited at the fifth phosphate. UvrABC nuclease incision kinetics revealed a hierarchy of specificity. The intercalative cis isomers were incised more efficiently than the corresponding trans isomers which lie in the minor groove. The (+) enantiomers were incised more efficiently than the (-) form for both cis and trans isomers. These observations reveal that UvrABC nuclease recognition and incision are directly influenced by the conformation of the DNA adduct. 59 refs., 10 figs.
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- FG02-86ER60405
- OSTI ID:
- 416002
- Journal Information:
- Biochemistry (Eaton), Journal Name: Biochemistry (Eaton) Journal Issue: 41 Vol. 34; ISSN 0006-2960; ISSN BICHAW
- Country of Publication:
- United States
- Language:
- English
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