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, Michael D. Wyatt, Mark A. Berg
2005 Biophysical Journal  
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 85fold reduction in
more » ... nding 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.
doi:10.1529/biophysj.105.062695 pmid:16199493 pmcid:PMC1366978 fatcat:2nxpsbucqncfxbgdjrut57ywbe