Mechanical Manipulations of Single Molecules of DNA, Proteinand their Complexes

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9631153 Bustamante This application describes a series of experiments designed to investigate the response of DNA, proteins, and their complexes when subjected to mechanical stress. These studies are a continuation of previous effects by this laboratory to employ methods such as Optical Tweezers and Scanning Force Microscopy (SFM) to investigate the mechanical response of single molecules. The proposed studies are divided into three main groups: i) Studies of the elastic response of single dsDNA molecules; ii) Studies of the elastic response of RecA-DNA filaments; and iii) Studies of the mechanical denaturation of single molecules of T4 lysozyme and titin. %%% The results of these studies will provide completely new insights into the nature and magnitude of the intramolecular forces that hold together biological structures. In fact, previously published work on the elasticity of single dsDNA molecules has already resulted in the characterization of three distinctive regimes of DNA elasticity. These three regimes are likely to play different roles in the process of recombination in vivo. The experiments proposed here will extend these studies to directly investigate the mechanical properties of the recombinant filaments and the denaturation of single protein molecules. ***