Ted Laurence, Ph.D.
Chemistry, Materials, and Life Sciences Directorate
Lawrence Livermore National Laboratory
 
 
Single-molecule fluorescence spectroscopy provides a new way to understand protein dynamics: isolate the protein and watch that one molecule for a long period of time.  By watching proteins in action one at a time, we are able to obtain the previously hidden dynamical information necessary to understand the mechanisms and limitations of protein machines.  The goal of our description of protein machines is to move beyond cartoon depiction of protein action.  Rather than simply determining what the moving parts are, we want to know how much friction there is between the moving parts, how much power is supplied by a chemical reaction, how does binding of another protein affect the action of the protein fluctuations? The details of the motion of the polIII β-subunit DNA sliding clamp (a.k.a. “β-clamp”) are the focus of this combined experimental and computational study.  By encircling the DNA, the DNA sliding clamp proteins remain stably attached to DNA while able to traverse very long distances along a DNA double helix. Present in all cellular (and some viral) forms of life, DNA sliding clamps attach to polymerases and allow the polymerase assembly to achieve rapid, processive, coordinated replication of both strands of DNA. Our study concerns the possible role and nature of molecular friction between the clamp and the DNA. Collaborators performed and analyzed molecular dynamics simulations to identify key protein-DNA interactions.  I have performed single-molecule measurements on a fluorescently labeled β-clamp loaded onto freely diffusing plasmids annealed with fluorescently labeled primers of up to 90 bases.  Comparing experiments using the primers of different lengths, we find that the sliding clamp stays at the 3’ end, ready for replication.  These results may have important implications for the initiation of lagging strand DNA replication. 
 
This work was performed under the auspices of the U.S. Department of Energy by the University of California, Lawrence Livermore National Laboratory under Contract No. W-7405-Eng-48.
 
 Please contact Thomas Huser (trhuser@ucdavis.edu) if you would like to meet with the speaker.

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