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

¹ University of South Carolina, Columbia, South Carolina 29208
² Presbyterian College, Clinton, South Carolina 29325
³ The Ohio State University, Columbus, Ohio 43210
⁴ National Institute of Aging, Baltimore, MD 21224

^* To whom correspondence should be addressed. E-mail: berg{at}mail.chem.sc.edu.

Submitted on March 11, 2005
Revised on April 24, 2005
Accepted on 1 September 2005

	Abstract

Synthetic oligonucleotides with a fluorescent coumarin group replacing a base-pair have been used in recent time-resolved Stokes-shift experiments to measure DNA dynamics on the femtosecond to nanosecond time scales. Here, we show that the APE1 endonuclease cleaves such a modified oligonucleotide at the abasic site opposite the coumarin with only a four-fold 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. However, a difference in the absolute Stokes shift at 40 ps suggests that differences might occur at shorter times.

Key Words: Stokes shift, abasic site, picosecond, solvation, solvatochromism