Interaction of the UvrABC Nuclease System with a DNA Duplex Containing a Single Stereoisomer of dG-(+)- or dG-(-)-anti-BPDE

Yue Zou, Tong Ming Liu, Nicholas E. Geacintov, Bennett Van Houten

Research output: Contribution to journalArticle

Abstract

Oligonucleotides containing site-specifically-modified N2-guanine (+)-trans-, (-)-trans-, (+)-cis-, and (-)-ds-BPDE adducts were ligated into 50-base-pair DNA fragments. These substrates were used in reactions with the Escherichia coli UvrABC nuclease system. The interaction of the UvrA2 and UvrA2B 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 UvrA2B appeared to interact very similarly with all four substrates. Binding of UvrA2 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’ and the fifth, sixth, or seventh phosphate 3’ to the modified guanine. The variation of the 3’ incision site was linked to the stereochemistry and orientation of the BPDE adduct. For example, the 3’ incision of the 50-bp duplex containing (-)-trans-BPDE-N2- 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.

Original languageEnglish (US)
Pages (from-to)13582-13593
Number of pages12
JournalBiochemistry
Volume34
Issue number41
DOIs
StatePublished - Oct 1995

ASJC Scopus subject areas

  • Biochemistry

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