Investigating the Nucleation and Extension Rates of E.coli and Deinococcus RecA Along Duplex DNA

Abstract

RecA is a protein which promotes the exchange between two homologous DNA molecules in homologous recombination process. When individual RecA molecules assemble on DNA, the DNA is stretched and underwound to form a nucleoprotein filament with its rigidity and end-to-end length increased. We have developed single-molecule tethered particle motion (TPM) experiments to study the assembly dynamics of RecA proteins on individual duplex DNA molecules. The TPM method is capable of measuring the changes in DNA length by observing the bead's Brownian motion, thus allowing us to monitor RecA nucleation and extension in real-time. Using much shorter DNA (a few hundreds basepairs), TPM experiments offer improved sensitivity, since the DNA length change can be readily detected as soon as a few RecA bounded to duplex DNA molecules. Our experiments indicated a faster nucleation rate compared to the previous reports (Galletto et al., 2006). Moreover, we have compared the nucleation and extension rates of E. coli RecA with the RecA from Deinococcus radiodurans, UV-resistant bacteria, under different nucleotide states, ATP and ATPγS. Deinococcus radiodurans RecA are found to nucleate faster (∼1.6×10-2 bp-1min-1) but extend slower (∼0.3-1.5 RecA/sec) under ATP.This difference reflects the physiological role of Dr. RecA when extensive UV-damaged DNA molecules are present.