Search this Site

Print this page
RSS

March 11, 2004

Thomas T. Perkins
Associate JILA Fellow
Adjoint Assistant Professor of MCDB
JILA, NIST-University of Colorado at Boulder

"Searching DNA with optical tweezers, one molecule at a time

Optical tweezers have become an integral tool for studying the mechanochemical cycle of motor proteins kinesin and myosin. These advances have now been applied to the study of enzymes that act on RNA and DNA, in particular RNA and DNA polymerase. Our research has focused on processive helicases and exonucleases, some of which depend on ATP hydrolysis and some of which do not. For ATP-independent exonucleases, the free energy available to drive the exonucleic reaction is unknown, but it is presumed to be significantly lower than for ATP-dependent enzymes. The speeds of digestion were nearly constant, around 5 nm/s (15 nt/s), interspersed with numerous pauses of variable duration. Long pauses tended to occur at stereotyped locations, and were strand-specific as well as and sequence-dependent. Sequencing gels were used to identify the DNA segment carrying the strongest pause, GGCGATTCT. A statistical analysis correlating single-molecule dwell positions with sequence independently identified the motif GGCGA.

Additionally, we studied RecBCD, a processive, ATP dependent DNA-based motor enzyme with both helicase and nuclease activities. Fine-scale motion was smooth down to a detection limit of 2 nm, implying a unitary step size below 6 bp. Interestingly, episodes of constant-velocity motion over hundreds to thousands of base pairs were punctuated by abrupt switches to a different speed or by spontaneous pauses of mean length 3 s . RecBCD occasionally reversed direction, sliding backwards along DNA. Backsliding could be halted by reducing the force, after which forward motion sometimes resumed, often after a delay. Our observations show that RecBCD-DNA complexes can exist in multiple, functionally distinct states that persist for many catalytic turnovers: such states may help tune enzyme activity for various biological functions.