Nanoscale Dynamics of Protein-DNA Complexes

Abstract

Time lapse AFM is the technique with a great potential for molecular biophysics. Advances in the sample preparation techniques for AFM coupled with emerging of high-speed AFM (HS AFM) instrumentation led to the progress in the study of a number of protein-DNA complexes. The mechanism by which site-specific binding proteins accomplish the search of cognate sites on DNA is one of the important ones. The problem is more severe if the systems requiring interaction with two specific DNA sites (such as site-specific recombinases) are concerned. EcoRII restriction enzyme belongs to the family of such site-specific enzyme and with the use of HS AFM we were able to identify a novel process for the site search for this two-site binding protein. We have shown that EcoRII is able to bind to one site of DNA pulling another DNA duplex in the search of another site. The time-lapse AFM technique was also instrumental in understanding dynamics of nucleosomes. We demonstrated that nucleosomes are quite dynamic rather than static systems and answered a number of important questions related to their dynamics. Single-stranded DNA binding proteins are ubiquotous systems involved in DNA replication, recombination, transcription and various modifications of DNA. We have developed a novel approach for studies of ssDNA binding proteins with AFM in which a hybrid DNA substrate with desired structure and sequence is used. In such designs, ssDNA of a specific length is either attached to one of the ends of dsDNA or placed between the two DNA duplexes. Time-lapse HS AFM was applied to look at the DNA-protein interaction dynamics including dissociationa and association steps for both systems and A3G sliding along the DNA substrate.