A number of vital biological processes rely on fast and precise recognition of a specific DNA sequence (site) by a protein. How can a protein find its site on a long DNA molecule among 10 6 -10 9 decoy sites? Here, we present our recent studies of the protein-DNA search problem. Seminal biophysical works suggested that the protein-DNA search is facilitated by 1D diffusion of the protein along DNA (sliding). We present a simple framework to calculate the mean search time and focus on several new aspects of the process such as the roles of DNA sequence and protein conformational flexibility. We demonstrate that coupling of DNA recognition with conformational transition within the protein-DNA complex is essential for fast search. To approach the complexity of the in vivo environment, we examine how the search can proceed at realistic DNA concentrations and binding constants. We propose a new mechanism for local distance-dependent search that is likely essential in bacteria. Simulations of the search on tightly packed DNA and crowded DNA demonstrate that our theoretical framework can be extended to correctly predicts search time in such complicated environments. We relate our findings to a broad range of experiments and summarize the results of our recent singlemolecule studies of a eukaryotic protein (p53) sliding along DNA.

The tumor suppressor p53 slides along DNA while searching for its cognate site. Central to this process is the basic C-terminal domain, whose regulatory role and its coordination with the core DNAbinding domain is highly debated. Here we use single-molecule techniques to characterize the search process and disentangle the roles played by these two DNA-binding domains in the search process. We demonstrate that the C-terminal domain is capable of rapid translocation, while the core domain is unable to slide and instead hops along DNA. These findings are integrated into a model, in which the C-terminal domain mediates fast sliding of p53, while the core domain samples DNA by frequent dissociation and reassociation, allowing for rapid scanning of long DNA regions. The model further proposes how modifications of the C-terminal domain can activate "latent" p53 and reconciles seemingly contradictory data on the action of different domains and their coordination.recognition | response element | transcription factor

The p53 protein, a transcription factor of key importance in tumorigenesis, is suggested to diffuse one-dimensionally along DNA via its C-terminal domain, a process that is proposed to regulate gene activation both positively and negatively. There has been no direct observation of p53 moving along DNA, however, and little is known about the mechanism and rate of its translocation. Here, we use single-molecule techniques to visualize, in real time, the one-dimensional diffusion of p53 along DNA. The one-dimensional diffusion coefficient is measured to be close to the theoretical limit, indicative of movement along a free energy landscape with low activation barriers. We further investigate the mechanism of translocation and determine that p53 is capable of sliding--moving along DNA while in continuous contact with the duplex, rather than through a series of hops between nearby bases.

Recognition and binding of specific sites on DNA by proteins is central for many cellular functions such as transcription, replication, and recombination. In the search for its target site, the DNA-associated protein is facing both thermodynamic and kinetic difficulties. The thermodynamic challenge lies in recognizing and tightly binding a cognate (specific) site among the billions of other (non-specific) sequences on the DNA. The kinetic difficulty lies in finding a cognate site in mere seconds amidst the crowded cellular environment that is filled with other DNA sequences and proteins. In this minireview, we will discuss the history of the DNA search problem, the theoretical background and the various experimental methods used to study the kinetics of proteins searching for target sites on DNA.

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...

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