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Effect of Protein Binding on Ultrafast DNA Dynamics: Characterization of a DNA:APE1 Complex

Sobhan Sen ^*, Nicole A. Paraggio ^*, Latha A. Gearheart , Ellen E. Connor , Ala Issa , Robert S. Coleman , David M. Wilson, 3rd ^¶, Michael D. Wyatt  and Mark A. Berg ^*

^* Department of Chemistry and Biochemistry and Department of Basic Pharmaceutical Sciences, University of South Carolina, Columbia, South Carolina; Department of Chemistry, Presbyterian College, Clinton, South Carolina; Department of Chemistry, The Ohio State University, Columbus, Ohio; and ^¶ Laboratory of Molecular Gerontology, National Institute of Aging, Baltimore, Maryland

Correspondence: Address reprint requests to Mark A. Berg, Dept. of Chemistry and Biochemistry, University of South Carolina, Columbia, SC 29208. Tel.: 803-777-1514; E-mail: berg{at}mail.chem.sc.edu.

Synthetic oligonucleotides with a fluorescent coumarin group replacing a basepair have been used in recent time-resolved Stokes-shift experiments to measure DNA dynamics on the femtosecond to nanosecond timescales. Here, we show that the APE1 endonuclease cleaves such a modified oligonucleotide at the abasic site opposite the coumarin with only a fourfold reduction in rate. In addition, a noncatalytic mutant (D210N) binds tightly to the same oligonucleotide, albeit with an 85-fold reduction in binding constant relative to a native oligonucleotide containing a guanine opposite the abasic site. Thus, the modified oligonucleotide retains substantial biological activity and serves as a useful model of native DNA. In the complex of the coumarin-containing oligonucleotide and the noncatalytic APE1, the dye's absorption spectrum is shifted relative to its spectrum in either water or within the unbound oligonucleotide. Thus the dye occupies a site within the DNA:protein complex. This result is consistent with modeling, which shows that the complex accommodates coumarin at the site of the orphaned base with little distortion of the native structure. Stokes-shift measurements of the complex show surprisingly little change in the dynamics within the 40 ps–40 ns time range.