DEVELOPING LABEL-FREE SINGLE-MOLECULE ASSAYS TO ENABLE PRECISE QUANTIFICATION OF DYNAMICS IN DNA-PROCESSING PROTEINS

Abstract

A class of protein called helicases play the pivotal roles behind the transitions between double-stranded and single-stranded DNA, by actively engaging DNA unwinding, rewinding, and translocation functions. However, the molecular mechanisms behind remain largely elusive, and the lack of accurate probing assays is the major obstacle. To cater to this problem, two fluorescent-label-free single-molecule translocation assays were designed based on our ultra-stable magnetic tweezers. Using these assays, precise quantification of Saccharomyces cerevisiae Pif1 activities were achieved. Two translocation modes of Pif1, dubbed as mobile translocation and loop-generation activity, were identified. The concentration of helicase was found to dictate the switch between the dual translocation modes. Our studies also revealed the same dual translocation modes being evident for another helicase UvrD. Together with the rewinding activity discovered on both helicases, our observations suggested that the properties more common than expected may be shared among helicases, implying a general molecular mechanism behind.