Real-Time Fluorescent Observation of DNA-Protein Interaction at the Single-molecule Level

Abstract

well-known DNA digestive enzyme, and the property has Single-molecule direct observations have been studied by been well studied by electrophoretic assay [7]. Exolll is a many researchers to reveal molecular behaviors usually 3'->5' dsDNA-specific exonuclease. Therefore, it is hidden in the ensemble and time averaging of bulk possible to digest from the blunt end, 3'-recessed end and experiments. In recent years, single-molecule analyses of nicking sites, but it is impossible to digest from the various DNA-protein interactions by using florescent 3 '-protruding end. The final product is single-stranded DNA techniques were performed. In this study, and 5'-P mononucleotides. Salt concentration and single-molecule DNA hydrolysis by exonuclease III and temperature both significantly affect ExollI activity. At X exonuclease were observed. We observed the DNA 23-28°C, the exonucleolytic digestion is processive, and digestion of individual stretched DNA molecules from under saturating enzyme concentrations at this temperature, the free end. The shortening of fluorescently stained 5' mononucleotides are removed at a rate of -100 DNA was recorded and analyzed. The sequentially nucleotides per minute (nt/min). At 37°C, the digestion is captured photographs demonstrate that the digested distributive, and digestion rate is -400 nt/min at saturating DNA molecule linearly shortened with reaction time. enzyme concentrations. These observations were based on a simple experimental In contrast, X exonuclease (Xexo) is involved in phage DNA setup. A relationship between a physical form and recombination and repair [8]. In the presence of Mg2' and a biochemical reaction is very interesting. Single-molecule 5'-terminal phosphate group, Xexo releases experiments should elucidate the mechanism of 5'-monodeoxyribonucleotides and produces non-hydrolyzed force-related DNA-protein interactions and provide single-stranded DNA by digesting the other strand of additional insight into these interactions in living cells.double-stranded DNA in the 5' -> 3' direction. It is reported that the substrate DNA is held by passing through the center