TCB Seminar

NANOSCALE GYMNASTICS OF PROTEIN-DNA COMPLEXES. TIME LAPSE HIGH SPEED AFM IMAGING

Professor Yuri Lyubchenko
Pharmaceutical Sciences
University of Nebraska Medical Center
Unknown Location

Monday, May 1, 2017
3:00 pm
3269 Beckman Institute

Abstract

Time lapse AFM is the technique with a great potential for single molecule biophysics. Advances in the sample preparation techniques for AFM coupled with unique capabilities of high-speed AFM (HS AFM) instrumentation led to the progress in the study of a number of protein-DNA complexes. A few topics will be covered. Single-stranded DNA binding proteins (SSBs) are ubiquotous systems involved in DNA replication, recombination, transcription and various modifications of DNA such as deamination (APOBEC proteins). The major challenge with structural studies of proteins working on ssDNA is the need in the unambigous identification of the complexes distinguishing them from uncomplexed proteins and non-specific complexes with double-stranded DNA (dsDNA). We have developed a novel hybrid DNA approach for AFM in which ssDNA of a specific length is attached to dsDNA. Time-lapse HS AFM was applied to visualize dynamics of DNA-protein interaction including dissociationa and association steps for SSB protein. 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 complex 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. The application of HS-AFM has led to the discovery a novel search mechanism in which EcoRII after binding to one site on the DNA template makes a loop and searches another site by pulling the DNA duplex through a second DNA binding site. The time-lapse AFM technique was also instrumental in characterization dynamics of nucleosomes. What is the range of dynamics of nucleosomes? Is a non-ATP dependent unwrapping of nucleosomes possible? What are the factors facilitating a large scale dynamics and unwrapping of nucleosomes? These questions were answered with the use of time-lapse AFM. Briefly, we demonstrated that nucleosomes are so dynamic that they are capable of spontaneous dissociation. The process is complex, so in addition to unwrapping, DNA is accessed by the nucleosome sliding.


Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198-6025, U. S. A.