Testing mechanisms of DNA sliding by architectural DNA-binding proteins: dynamics of single wild-type and mutant protein molecules in vitro and in vivo

Abstract

Architectural DNA-binding proteins (ADBPs) are abundant constituents of eukaryotic or bacterial chromosomes that bind DNA promiscuously and function in diverse DNA reactions. They generate large conformational changes in DNA upon binding yet can slide along DNA when searching for functional binding sites. Here we investigate the mechanism by which ADBPs diffuse on DNA by single-molecule analyses of mutant proteins rationally chosen to distinguish between rotation-coupled diffusion and DNA surface sliding after transient unbinding from the groove(s). The properties of yeast Nhp6A mutant proteins, combined with molecular dynamics simulations, suggest Nhp6A switches between two binding modes: a static state, in which the HMGB domain is bound within the minor groove with the DNA highly bent, and a mobile state, where the protein is traveling along the DNA surface by means of its flexible N-terminal basic arm. The behaviors of Fis mutants, a bacterial nucleoid-associated helix-turn-helix dimer, are best explained by mobile proteins unbinding from the major groove and diffusing along the DNA surface. Nhp6A, Fis, and bacterial HU are all near exclusively associated with the chromosome, as packaged within the bacterial nucleoid, and can be modeled by three diffusion modes where HU exhibits the fastest and Fis the slowest diffusion.

Single-Molecule Microscopy Meets Molecular Dynamics Simulations for Characterizing the Molecular Action of Proteins on DNA and in Liquid Condensates

“…This is also supported by the fact that the designed peptide targeting the CT domain suppresses the sliding of p53 along DNA (Kamagata et al, 2019). Similarly, the deletion of basic IDRs from the nucleoid protein Nhp6A significantly slows its sliding along DNA, which was also assessed using coarse-grained MD simulations (Kamagata et al, 2021b). Nhp6A in fast mode slides along DNA, contacting the basic IDR without interaction of the folded HMGB domain with DNA, while Nhp6A tightly binds to DNA using the folded HMGB domain in slow mode (Figure 1C).…”

“…It forms a tetramer and utilizes a folded core domain and a disordered C-terminal (CT) domain for DNA binding in a specific and nonspecific manner, respectively (Anderson et al, 1997). Fifty percent of gene mutations in tumor cells were found in p53, and (Itoh et al, 2018;Subekti et al, 2020;Kamagata et al, 2021b;Kamagata, 2021) with some modifications. Panel (E) was reprinted with permission from (Takada et al, 2015) Copyright 2015 American Chemical Society with some modifications.…”

“…For the latter, wedge residues in glycosylases (Phe residue in Fpg, Tyr residue in Nei, and Leu residue in Nth) have been identified to intercalate into DNA bases, increasing the sliding friction of these proteins (Dunn et al, 2011;Nelson et al, 2014). Similarly, removing intercalating residues in Nhp6A (Met and Phe residues) facilitates its sliding along DNA (Kamagata et al, 2021b). Furthermore, the Arg residue in the nucleoid protein Fis significantly suppresses its sliding dynamics (Kamagata et al, 2021b).…”

“…Similarly, removing intercalating residues in Nhp6A (Met and Phe residues) facilitates its sliding along DNA (Kamagata et al, 2021b). Furthermore, the Arg residue in the nucleoid protein Fis significantly suppresses its sliding dynamics (Kamagata et al, 2021b). Thus, the combination of single-molecule microscopy and MD simulation has clarified some molecular principles of the sliding dynamics of DNA-binding proteins.…”

Interactions of HMGB Proteins with the Genome and the Impact on Disease

“…We also discuss recent evidence characterizing HMGB protein interactions with G-quadruplex structures that form in promoters and at telomeres, as well as a growing body of data showing that different types of cellular RNA could be important regulatory partners for HMGB proteins. Although the focus of this review is mammalian HMGB proteins, it is worth noting that Saccharomyces cerevisiae Nhp6, a yeast counterpart to HMGB1/2, has been extensively investigated using structural, biochemical, and biophysical approaches, which provide important insight into how HMG boxes interact with DNA and chromatin [59][60][61][62].…”