Sequence-Dependent Pausing of Single Lambda Exonuclease Molecules
Thomas T. Perkins,1*
Ravindra V. Dalal,2
Paul G. Mitsis,3
Steven M. Block1,4
Lambda exonuclease processively degrades one strand of duplex
DNA, moving 5'-to-3' in an ATP-independent fashion. When examined
at the single-molecule level, the speeds of digestion were nearly
constant at 4 nanometers per second (12 nucleotides per second),
interspersed with pauses of variable duration. Long pauses,
occurring at stereotypical locations, were strand-specific and
sequence-dependent. Pause duration and probability varied widely.
The strongest pause, GGCGAT TCT, was identified by gel electrophoresis.
Correlating single-molecule dwell positions with sequence independently
identified the motif GGCGA. This sequence is found in the left
lambda cohesive end, where exonuclease inhibition may contribute
to the reduced recombination efficiency at that end.
2 Department of Physics, Stanford University, Stanford, CA 94305, USA.
4 Department of Applied Physics, Stanford University, Stanford, CA 94305, USA.
3 Praelux, Inc., Lawrenceville, NJ 08646, USA.
* Present address: JILA, National Institute of Standards and Technology and University of Colorado, Boulder, CO80309–0440, USA.
Present address: Amersham Biosciences, 800 Centennial Road, Piscataway, NJ 08855, USA.
To whom correspondence should be addressed. E-mail: tperkins{at}jila.colorado.edu
