Thermodynamics and kinetics of DNA-protein interactions from single molecule force spectroscopy

Abstract

The process of replication requires the cooperation of many proteins which associate with each other at the replication fork to form a highly efficient replication machine. Bacterium E. coli and bacteriophages that infect it (T4 and T7) have been used extensively in molecular biology research and provide excellent model systems for analyzing the DNA replication. In this work we use single DNA molecule stretching to investigate the degree of alteration in the structure and stability of DNA in the presence of DNA binding proteins which help us to quantify thermodynamics and kinetics of protein-protein and protein-nucleic acid interactions and obtain new insights into the function of proteins in these specific biological system. Because the nature of the overstretching transition in DNA stretching experiments continues to generate controversy we have undertaken studies of DNA stretching in the presence of glyoxal to solve this dilemma which brought new evidence in favor of force-induced melting theory against the alternative S-DNA model. One of the classic paradigms of single-stranded DNA binding proteins is bacteriophage T7 gene 2.5 protein (gp2.5), known to have essential roles in DNA replication and recombination in phage-infected cells by binding preferentially to single-stranded DNA and establishing electrostatic interactions with other proteins, recruiting them at the site of the ssDNA and regulating their activity. Varying solution conditions and the pulling dynamics, we could obtain binding affinities to single- and double-stranded DNA for gp2.5 and its deletion mutant lacking 26 C-terminal residues, gp2.5-delta26C, over a range of salt concentration not available to ensemble studies. We also obtained rate of cluster growth which is analogous to the growth of clusters that occurs at a replication fork as the helicase unwinds the double helix in vivo. We proposed a model to explain proteins' structure-function relationship, and showed that dimeric gp2.5 must dissociate prior to binding to DNA, a dissociation that consists of a weak non-electrostatic and a strong electrostatic component. The multisubunit enzyme DNA polymerase III holoenzyme (pol III) is responsible for duplicating the E. coli chromosomal DNA. By purifying fragments of the protein that encompass the putative OB-fold domain, and then characterizing those fragments for their DNA binding activity using single molecule DNA stretching experiments, we showed that a subunit has an affinity for both double and single stranded DNA. However, our results demonstrate that the single-stranded DNA binding component appears to be passive, as the protein does not facilitate melting, but binds instead to regions already separated by force, stabilizing the single-stranded form of DNA. By studying the constructs of segments of the a subunit we showed that the N-terminal region is responsible for dsDNA stabilization, while the C-terminal region binds to melted DNA suggesting that this domain may interact with ssDNA created during the DNA replication process. Bacteriophage T4 gene 59 (gp59) protein is a replication-recombination mediator protein that stimulates the activities of the helicase enzyme by promoting its loading onto gene 32 protein (gp32)-saturated single-stranded DNA binding sites. We characterized the interactions of wild type protein gp59 and its site-directed mutant with defects in dsDNA binding (gp59R12A) with stretched DNA. We showed that gp59 binds more strongly to double-stranded DNA and determined the equilibrium binding constant to dsDNA as a function of salt concentration. We also examined the effects of gp59 on the helix-destabilization capabilities of gp32 and its fragment, *I. Our results demonstrate that gp59 is capable of strongly destabilizing both gp32-DNA and *I-DNA interactions. This result confirms previous assumption that the local weakening of gp32-ssDNA binding is a requirement for helicase loading at that site.