Transições conformacionais no DNA polimerase I revelado por uma molécula FRET

terça-feira, janeiro 19, 2010


Conformational transitions in DNA polymerase I revealed by single-molecule FRET

  1. Yusdi Santoso a,1
  2. Catherine M. Joyce b,1,2
  3. Olga Potapova b,
  4. Ludovic Le Reste a
  5. Johannes Hohlbeina
  6. Joseph P. Torella a,
  7. Nigel D. F. Grindley b, and 
  8. Achillefs N. Kapanidis a2
    Author Affiliations

    1. aBiological Physics Research Group, Department of Physics, Clarendon Laboratory, University of Oxford, Oxford, OX1 3PU, United Kingdom,

      bDepartment of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520



    2.  1Y.S. and C.M.J. contributed equally to this work.

    3. Edited by Stephen J Benkovic, The Pennsylvania State University, University Park, PA, and approved November 18, 2009 (received for review October 3, 2009)

    1. Abstract
      The remarkable fidelity of most DNA polymerases depends on a series of early steps in the reaction pathway which allow the selection of the correct nucleotide substrate, while excluding all incorrect ones, before the enzyme is committed to the chemical step of nucleotide incorporation. The conformational transitions that are involved in these early steps are detectable with a variety of fluorescence assays and include the fingers-closing transition that has been characterized in structural studies. Using DNA polymerase I (Klenow fragment) labeled with both donor and acceptor fluorophores, we have employed single-molecule fluorescence resonance energy transfer to study the polymerase conformational transitions that precede nucleotide addition. Our experiments clearly distinguish the open and closed conformations that predominate in Pol-DNA and Pol-DNA-dNTP complexes, respectively. By contrast, the unliganded polymerase shows a broad distribution of FRET values, indicating a high degree of conformational flexibility in the protein in the absence of its substrates; such flexibility was not anticipated on the basis of the available crystallographic structures. Real-time observation of conformational dynamics showed that most of the unliganded polymerase molecules sample the open and closed conformations in the millisecond timescale. Ternary complexes formed in the presence of mismatched dNTPs or complementary ribonucleotides show unique FRET species, which we suggest are relevant to kinetic checkpoints that discriminate against these incorrect substrates.


      • alternating-laser excitation
      •  
      • conformational dynamics
      • fidelity checkpoints
      •  
      • Klenow fragment
      •  
      • fingers-closing
        Footnotes
        • 2To whom correspondence may be addressed. E-mail:a.kapanidis1@physics.ox.ac.uk or catherine.joyce@yale.edu

        • Author contributions: C.M.J., N.D.F.G., and A.N.K. designed research; Y.S., C.M.J., O.P., and L.L.R. performed research; Y.S., C.M.J., O.P., J.H., and J.P.T. contributed new reagents/analytic tools; Y.S., C.M.J., J.H., J.P.T., and A.N.K. analyzed data; and Y.S., C.M.J., N.D.F.G., and A.N.K. wrote the paper.
          The authors declare no conflict of interest.
          This article is a PNAS Direct Submission.
          This article contains supporting information online at www.pnas.org/cgi/content/full/0910909107/DCSupplemental.
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