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.
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