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  • Effect of protein binding on ultrafast DNA dynamics: characterization of a DNA:APE1 complex.

Effect of protein binding on ultrafast DNA dynamics: characterization of a DNA:APE1 complex.

Biophysical journal (2005-10-04)
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
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.

MATERIALS
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Product Description

Sigma-Aldrich
Aminopeptidase I from Streptomyces griseus, lyophilized powder, ≥200 units/mg protein