Hydrophobic forces dominate the thermodynamic characteristics of UvrA-DNA damage interactions

Yue Zou, Heather Bassett, Randall Walker, Adriana Bishop, Shantu Amin, Nicholas E. Geacintov, Bennett Van Houten

Research output: Contribution to journalArticlepeer-review


The Escherichia coli DNA repair proteins UvrA, UvrB and UvrC work together to recognize and incise DNA damage during the process of nucleotide excision repair (NER). To gain an understanding of the damage recognition properties of UvrA, we have used fluorescence spectroscopy to study the thermodynamics of its interaction with a defined DNA substrate containing a benzo[a]pyrene diol epoxide (BPDE) adduct. Oligonucleotides containing a single site-specifically modified N2-guanine (+)-trans-, (-)-trans-, (+)-cis-, or (-)-cis-BPDE adducts were ligated into 50-base-pair DNA fragments. All four stereoisomers of DNA-BPDE adducts show an excitation maximum at 350 nm and an emission maximum around 380 to 385 nm. Binding of UvrA to the BPDE-DNA adducts results in a five to sevenfold fluorescence enhancement. Titration of the BPDE-adducted DNA with UvrA was used to generate binding isotherms. The equilibrium dissociation constants for UvrA binding to (+)-trans-, (-)-trans-, (+)-cis-, and (-)-cis- BPDE adduct were: 7.4 ± 1.9, 15.8 ± 5.4, 11.3 ± 2.7 and 22.4 ± 2.0 nM, respectively. There was a large negative change in heat capacity ΔC(p,obs)(o), (-3.3 kcal mol-1 K(-l)) accompanied by a relatively unchanged ΔG(obs)(o) with temperature. Furthermore, varying the concentration of KC1 showed that the number of ions released upon formation of UvrA-DNA complex is about 3.4, a relatively small value compared to the contact size of UvrA with the substrate. These data suggest that hydrophobic interactions are an important driving force for UvrA binding to BPDE-damaged DNA.

Original languageEnglish (US)
Pages (from-to)107-119
Number of pages13
JournalJournal of Molecular Biology
Issue number1
StatePublished - Aug 7 1998

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology


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