· W. Allen, P.J. Rothwell and G. Waksman.
An intramolecular FRET system monitors fingers subdomain opening in Klentaq11
Protein Science, In Press (2008).
A major goal of polymerase research is to determine the mechanism
through which a nucleotide complementary to a templating DNA base is selected
and delivered to the polymerase active site. Structural evidence suggests a
large open-to-closed conformational change affecting the fingers subdomain as
being crucial to the process. We previously designed a FRET system capable of
measuring the rate of fingers subdomain closure in the presence of correct
nucleotide [Rothwell et al. (2005) Molecular Cell, 19:345-355; Rothwell and
Waksman (2007) J. Biol. Chem. 282, 28884-28892]. However this FRET system was
limited in that it could not directly measure the rate of fingers subdomain
opening by FRET after polymerisation, or in the absence of DNA. Here we report
the development of a new system capable of measuring both fingers subdomain
closure and reopening by FRET, and show that the rate of fingers subdomain
opening is limited only by the rate of polymerization. We anticipate that this
system will scale down to the single molecule level, allowing measurement of
fingers subdomain movements in the presence of incorrect nucleotide, and in the
absence of DNA.