Effect of protein binding on ultrafast DNA dynamics: characterization of a DNA:APE1 complex |
| |
Authors: | Sen Sobhan Paraggio Nicole A Gearheart Latha A Connor Ellen E Issa Ala Coleman Robert S Wilson David M Wyatt Michael D Berg Mark A |
| |
Institution: | Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina 29208, USA. |
| |
Abstract: | 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. |
| |
Keywords: | |
本文献已被 ScienceDirect PubMed 等数据库收录! |
|