Effect of protein binding on ultrafast DNA dynamics: characterization of a DNA:APE1 complex.
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.
Sen
Sobhan
S
Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina 29208, USA.
Paraggio
Nicole A
NA
Gearheart
Latha A
LA
Connor
Ellen E
EE
Issa
Ala
A
Coleman
Robert S
RS
Wilson
David M
DM
Wyatt
Michael D
MD
Berg
Mark A
MA
D000626:Aminopeptidases / Q000737:chemistry*; D001665:Binding Sites; D003198:Computer Simulation; D004247:DNA / Q000737:chemistry* / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008968:Molecular Conformation; D011485:Protein Binding; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors
D004268:DNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D000626:Aminopeptidases; C019689:APE1 protein, S cerevisiae