1 research outputs found
Restriction Enzyme Ecl18kI-Induced DNA Looping Dynamics by Single-Molecule FRET
Many
type II restriction endonucleases require binding of two copies of
a recognition site for efficient DNA cleavage. Simultaneous interaction
of the enzyme with two DNA sites results in DNA loop formation. It
was demonstrated with the tethered particle motion technique that
such looping is a dynamic process where a DNA loop is repeatedly formed
and disrupted. Here we use a better and in the context of protein-induced
DNA looping virtually unexploited strategy of single-molecule Förster
resonance energy transfer of surface immobilized biomolecules to quantitatively
study the dynamics of Ecl18kI endonuclease-induced DNA looping and
determine the rate constants of loop formation and disruption. We
show that two DNA-bound Ecl18kI dimers efficiently form a bridging
tetramer looping out intervening DNA with a rate that is only a few
orders of magnitude lower than the diffusion limited rate. On the
other hand, the existence of Ecl18kI tetramer is only transient, and
the loop is rapidly disrupted within about 1 s