Center for Functional Nanomaterials Seminar

Recent years have witnessed the emergence of an entirely new field of science, often referred to as single-molecule (SM) biology. Atomic force microscopy (AFM) was used to understand the methylation-dependent chromatin structure. We performed AFM studies of fibers isolated from cultured cells containing normal or elevated levels of m5C. Chromatin fibers were reconstituted on control or methylated DNA templates in the presence or absence of linker histones. A strong dependence of the rate of assembly on the exerted force was observed, with complete inhibition of assembly at forces exceeding 10 pN.
A single-molecule fluorescence analysis was applied to study the dynamics of synaptic and presynaptic DNA-protein complexes (binding of two DNA and one DNA duplex, respectively). In this approach, the protein was tethered to a surface, allowing a freely diffusing fluorescently labeled DNA to bind to the protein, thus forming a presynaptic complex. The duration of fluorescence burst is the measure of the characteristic lifetime of the complex. The synaptic complex formation by the differently labeled duplexes was detected by the fluorescence resonance energy transfer approach (FRET). Single pair FRET was also used to study Holliday junction, which is a central intermediate in various genetic processes including homologous, site-specific recombination and DNA replication. The We have shown that insertion of a GC pair into the middle of the AT tract, it resides at this position much longer time in comparison to the folding at AT pairs, when the junction folds at this position. Two contiguous GC pairs do not block migration as well and generally manifest the same effect as one GC pair--the junction, when it folds, resides at these positions for a relatively long time. DNA sequence modulates the overall stochastic process of the junction dynamics and branch migration by the variability of the time that the junction dwells before making a migration hop.