Significance of Knotted Structures for Function of Proteins and Nucleic Acids - September 17-21, 2014

Significance of Knotted Structures for Function of Proteins and Nucleic Acids

Saturday Abstracts

Unveiling the Molecular Trajectory during Binding Site Recognition by DNA-bending Proteins Anjum Ansari . University of Illinois at Chicago, Chicago, USA. Many proteins that bind to specific sites on DNA locate these sites by first binding to nonspecific sites on DNA and then scanning nearby sites for their target binding site. Characterizing the rates and mechanism by which proteins switch from nonspecific to specific binding has been a challenge in the field. Here we demonstrate how the enhanced sensitivity and time-resolution of laser temperature-jump (T-jump) allows us to capture the kinetics of molecular rearrangements during binding site recognition by two classes of proteins: (1) Integration Host Factor (IHF), a eubacterial architectural protein involved in chromosomal compaction and DNA recombination, which recognizes specific DNA sites and bends them into sharp U-turns, and (2) XPC/Rad4, a DNA damage repair protein that initiates nucleotide excision repair by recognizing diverse DNA lesions caused by environmental insults. We use T-jump to perturb the protein-DNA complex and time-resolved fluorescence measurements to monitor the dynamics of DNA bending, unwinding, and nucleotide-flipping upon protein binding. In the two systems: IHF bound to one of its cognate sites on DNA and XPC bound to DNA containing a model (3-bp mismatch) lesion, we resolve relaxation kinetics occurring over two distinct timescales. A rapid (~100 microseconds) phase, which is found to be independent of the DNA sequence, is attributed to nonspecific “interrogation” of the DNA binding sites by the protein, while a slower (~10 milliseconds) phase is attributed to the ultimate recognition step to form the specific complex. These results represent the first observation of an apparent two-step (interrogation then recognition) process that bridges the gap between relatively slow (> ms) recognition of target sites and relatively rapid (< ms) one-dimensional diffusion of proteins scanning the DNA.

- 38 -