Direct imaging of single UvrD helicasedynamics on long single-stranded DNA

摘要

Fluorescence imaging of single-protein dynamics on DNA has been largely limited todouble-stranded DNA or short single-stranded DNA. We have developed a hybrid approachfor observing single proteins moving on laterally stretched kilobase-sized ssDNA. Here weprobed the single-stranded DNA translocase activity of Escherichia coli UvrD by singlefluorophore tracking, while monitoring DNA unwinding activity with optical tweezers tocapture the entire sequence of protein binding, single-stranded DNA translocationand multiple pathways of unwinding initiation. The results directly demonstrate that theUvrD monomer is a highly processive single-stranded DNA translocase that is stopped by adouble-stranded DNA, whereas two monomers are required to unwind DNA to a detectabledegree. The single-stranded DNA translocation rate does not depend on the force appliedand displays a remarkable homogeneity, whereas the unwinding rate shows significantheterogeneity. These findings demonstrate that UvrD assembly state regulates its DNAhelicase activity with functional implications for its stepping mechanism, and also reveal apreviously unappreciated complexity in the active species during unwinding.