Sequence-dependent sliding kinetics of p53

Abstract

Proper timing of gene expression requires that transcription factors (TFs) efficiently locate and bind their target sites within a genome. Theoretical studies have long proposed that one-dimensional sliding along DNA while simultaneously reading its sequence can accelerate TF's location of target sites. Sliding by prokaryotic and eukaryotic TFs were subsequently observed. More recent theoretical investigations have argued that simultaneous reading and sliding is not possible for TFs without their possessing at least two DNA-binding modes. The tumor suppressor p53 has been shown to slide on DNA, and recent experiments have offered structural and single molecule support for a two-mode model for the protein.If the model is applicable to p53, then the requirement that TFs be able to read while sliding implies that noncognate sites will affect p53's mobility on DNA, which will thus be generally sequencedependent. Here, we confirm this prediction with single-molecule microscopy measurements of p53's local diffusivity on noncognate DNA. We show how a two-mode model accurately predicts the variation in local diffusivity, while a single-mode model does not. We further determine that the best model of sequence-specific binding energy includes terms for "hemi-specific" binding, with one dimer of tetrameric p53 binding specifically to a half-site and the other binding nonspecifically to noncognate DNA. Our work provides evidence that the recognition by p53 of its targets and the timing thereof can depend on its noncognate binding properties and its ability to change between multiple modes of binding, in addition to the much better-studied effects of cognate-site binding.protein-DNA interactions | protein-DNA search | promoter search | energy landscape | one-dimensional diffusion T umor suppressor p53 is known as the "guardian of the genome." The protein is mutated in more than 50% of cancers (1), and plays important roles in activating DNA-repair, cell-cycle arrest, and apoptosis. To prevent the replication of damaged DNA, damage-activated p53 must reach its target promoters sufficiently fast.In addition to its clinical importance, p53 is the first eukaryotic transcription factor (TF) directly observed to undergo one-dimensional (1D) diffusion on DNA (2). This 1D sliding has long been proposed to facilitate search by DNA-binding proteins (DBPs) (3, 4) and was characterized by single-molecule experiments for RNA polymerase (5, 6), mitochondrial repair enzymes (7), lac repressor (8, 9), and repair complex Msh2-Msh6 (10) (see ref. 11 for review). Sliding of p53 was first demonstrated by bulk biochemical experiments and was shown to play a role in p53's activation of target genes (12, 13). Several theoretical and experimental studies (7,8,14,15) have shown that despite a vast excess of accessible DNA (10 7 -10 9 bp) to which DBPs have nonspecific affinity, their search process can be efficient if they alternate rounds of 1D sliding while bound nonspecifically with rounds of three-dimensional (3D) diffusion between different sec...

On the influence of protein-DNA register during homologous recombination

“…2C). One-dimensional diffusion of proteins on both ssDNA and dsDNA is now a well-documented phenomenon, [23][24][25][26][27][28][29][30][31] and we have previously observed 1D diffusion of human Rad51 along dsDNA, 32 supporting the notion that Rad51/RecA monomers within the presynaptic complex might also be capable of sliding at least short distances along ssDNA. The ability of Rad51/RecA to slide just §1-nucleotide would ensure optimal alignment of DNA triplet sequences during the early stages of homology recognition and strand exchange.…”

Structural visualization of the p53/RNA polymerase II assembly

“…Perhaps when the p53/RNA Pol II complex encounters a response element, association with additional p53 oligomers may help it to loop and bind the core promoter in a hemispecific manner via contacts within the PIC components. In support of this idea, a previous study suggests that one dimer of p53 occupies the response element, while the other dimer of the tetramer can bind nonspecific DNA (Leith et al 2012). Additionally, Pol II recruitment could be mediated by p53's interactions with other PIC components such as TFIID and Mediator.…”

Twist-open mechanism of DNA damage recognition by the Rad4/XPC nucleotide excision repair complex

“…It has been proposed that a conformational switch between a rapidly diffusing "search" mode and a more tightly bound "recognition" mode is needed for a protein to identify a potential target site without losing overall speed (23)(24)(25)(26). Consistent with these arguments, alternative binding modes of various proteins interacting with nonspecific DNA have been observed (27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37), and have also been inferred from singlemolecule studies of proteins diffusing on DNA (13,(38)(39)(40). Microsecond-to-millisecond conformational fluctuations have been reported in proteins nonspecifically bound to DNA in a few systems amenable to NMR (28,32,36).…”