RESEARCH Areas

Forces associated with DNA helicase translocation
and DNA unwinding

DNA helicases convert chemical energy into the mechanical forces required to separate DNA strands and to translocate, either on dsDNA or on the unwound single strands of DNA. Individual steps in the catalytic cycle (e.g., the motion of domains relative to one another or perhaps ATP hydrolysis product release) may be subject to forces applied to the enzyme.
Atomic force microscopy (AFM) was originally designed for the study of surface topography. We use the AFM to monitor forces for DNA helicases in action. AFM has been used to measure a variety of forces ranging from ~ 10 - 220,00 pN in size and in addition, has the sensitivity to measure angstrom-scale, millisecond events. It is anticipated that the magnitude of the forces for DNA helicases should fall within this range, as previous measurement of stall forces for motors ranged from 5-7 pN for kinesin to 35 pN for RNA polymerase. A previous study demonstrating the ability of a DNA helicase to disrupt the biotin-streptavidin interaction suggests that for DNA helicases stall forces may be even larger.
Force measurements are currently focusing on a Superfamliy I DNA helicase, the RecBCD enzyme of E. coli. RecBCD enzyme has been well studied mechanistically making it amenable to this type of study. This enzyme has a translocation step size of 23 nt, an unwinding step size of 4-5 bp and hydrolyzes 2-3 ATP molecules per bp of dsDNA unwound. Subsequent experiments will focus on a Superfamily II enzyme, the Type I restriction enzyme, EcoR124I.