Following the Motions of a DNA Helicase on DNA in Real Time

Abstract

Using several experimental approaches, we are investigating the dynamics of individual PcrA DNA helicase interactions on DNA templates. Bulk biophysical and biochemical measurements are done in parallel to ascertain the viability and integrity of the protein and DNA substrates. For Pacman, we are investigating the relationship between the hydrolysis of ATP and the mechanical motion of the enzyme along a DNA substrate. We have designed a mutant PcrA with two cysteines for attachment of a pair of dyes to follow internal protein motions using single-pair fluorescence resonance energy transfer (spFRET). For Ring-a-bell we are following the interaction of PcrA with replication protein RepC. Alone, PcrA is typically capable of translocating only ∼80 bp before dissociating from a DNA template. With RepC, PcrA can achieve rolling circle replication of thousands of bp on a plasmid. Should the two proteins be bound together, they should create specific low and high spFRET signals as they translocate along a specifically labeled DNA substrate. And finally we are studying the interaction of PcrA and Holiday Junction DNA. Does PcrA separate the DNA from the center or from the ends of the DNA junction? We have upgraded our evanescent field fluorescence microscope to use alternating red (648 nm) and green (532 nm) lasers to illuminate the sample. We have added multiple syringes operating under computer control to follow experiments in real-time whereupon ATP is introduced into the liquid flow chamber. We have also upgraded our scanning confocal microscope to improve the alignment of the laser and the ease of use of the instrument.