Dynamics and mechanism of damage recognition by DNA-repair proteins

Abstract

Altered unwinding/bending fluctuations at DNA lesion sites are implicated as plausible mechanisms for damage sensing by DNA-repair proteins. These dynamics are expected to occur on similar timescales as one-dimensional (1D) diffusion of proteins on DNA if effective in stalling these proteins as they scan DNA. We examined the flexibility and dynamics of DNA oligomers containing 3 base pair (bp) mismatched sites specifically recognized in vitro by nucleotide excision repair protein Rad4 (yeast ortholog of mammalian XPC). With laser temperature-jump, we revealed the timescales on which Rad4 unwinds DNA to flip out nucleotides from within damaged sites (1,2). With fluorescence lifetime and correlation spectroscopies, we uncovered significant deviations from canonical B-DNA-like conformations and large-amplitude unwinding dynamics for mismatched constructs specifically recognized by Rad4, even in the absence of Rad4 (3,4). These studies are the first to visualize anomalous unwinding/bending fluctuations in mismatched DNA, on timescales that overlap with the <500 µs “stepping” times of repair proteins on DNA. Such “flexible hinge” dynamics at lesion sites could arrest a diffusing protein to facilitate damage interrogation and recognition. The above studies, like most others on DNA damage recognition mechanisms, were performed with short, torsionally relaxed DNA oligomers that do not reflect DNA topologies in our cells, where the DNA is largely bent and/or supercoiled. Looping and supercoiling are expected to have a profound impact on damage sensing. We have begun studies of DNA dynamics and Rad4 binding in the context of 126-bp DNA minicircles that mimic the bending strain present in nucleosomes. We find that DNA distortions at the 3-bp mismatched site are amplified in these minicircles and that Rad4 binding affinities increase by >100-fold. These studies indicate that much of what we have learned about DNA conformations, dynamics, and damage recognition from studies on short DNA oligomers must be revisited.