Download Nugget

KINETICS MEASUREMENTS OF THE WRAPPING AND UNWRAPPING OF SINGLE-STRANDED DNA AROUND SINGLE
STRAND BINDING PROTEIN
Anjum Ansari, Physics Department, University of Illinois at Chicago, Chicago, IL 60607
The E. coli SSB protein is a helix-destabilizing protein that binds with high affinity to ssDNA intermediates during replication,
recombination and DNA repair. It has been proposed that SSB, while bound to ssDNA, can translocate along the ssDNA via a
rolling mechansim. The protein cannot translocate any faster than the rate at which ssDNA wraps and unwraps from around
SSB. Thus, a mechanistic understanding requires knowledge of the wrapping/unwrapping rates. In vitro, SSB binds to ssDNA
in two or more distinct binding modes. In the fully wrapped (SSB)65 mode, the two ends of a ~70-nt strand are expected to be
in close contact. Thus, FRET measurements between donor and acceptor molecules attached at either end of the ~70-nt long
ssDNA strand provide a sensitive probe of the wrapping and unwrapping of ssDNA.
Previous stopped-flow kinetic studies of SSB binding to (dT)70 were unable
to resolve the bimolecular association step from the unimolecular
wrapping/unwrapping step. We report the first direct observation of the
wrapping/unwrapping kinetics, in response to a laser T-jump perturbation of
a pre-formed SSB-(dT)70 complex. Time-resolved measurements were
made on two sets of samples: (i) FRET measurements on SSB:Cy5-(dT)69Cy3 complex (Fig. 1a-c), and (ii) Trp fluorescence measurements on
SSB:(dT)70 (Fig. 1d-f). In both sets of measurements, biphasic kinetics
were observed. The fast phase, which occurs on a time-scale of tens of
microseconds, is concentration-independent (Fig. 1b,e) and reflects the
wrapping/unwrapping of ssDNA around the SSB tetramer. Analysis of the
slower binding phase (Fig. 1c,f), in combination with rates obtained from
stopped-flow studies (oen symbols in Fig. 1c,f), provides evidence for a
previously undetected, partially wrapped, intermediate along the pathway to
forming the (SSB)65 complex (P*D in Fig. 2). The wrapping/unwrapping
process observed on microseconds time-scale sets an upper limit on how
fast SSB can translocate along DNA during replication, recombination, and
repair.
This study represents a novel application of laser T-jump techniques to
probe protein-DNA interactions, and demonstrates the powerful capabilities
of this technique to probe the dynamics of protein-DNA complexes on
microseconds-to-milliseconds time-scale.