Watching Individual DNA Helicases and Motor Proteins Behaving and Misbehaving

Abstract

We can now watch individual proteins acting on single molecules of DNA. Using these approaches, we have been imaging the translocation of motor proteins, such as the ssDNA translocases and DNA helicases, RecBCD, RecQ, and Sgs1, and the dsDNA translocases and chromatin-remodeling enzymes, Rad54 and Tid1 proteins.The RecBCD enzyme comprises two motor subunits: RecB, a 3’→5’ SF1-helicase, and RecD, a 5’→3’ SF1-helicase. Although the mean unwinding rate of individual RecBCD enzyme molecules is close to ensemble measurements, the rates of individual enzyme molecules vary widely. Individual RecBCD molecules unwind DNA at constant rates, suggesting static, not dynamic, disorder. This apparent conformational heterogeneity is static on the experimental time scale of DNA unwinding. We discovered that transiently halting a single enzyme-DNA complex changes the rates of the RecBCD molecule. We will demonstrate that the behavior of individual RBCD enzymes manifests a basic tenet of the ergodic hypothesis, suggesting that ligand binding kinetically traps a single conformer that is capable of redistributing to all other states within the population of molecules at equilibrium.Using TIRF microscopy, the helicase activity of RecQ was visualized on single molecules of DNA using a fluorescent ssDNA sensor. By monitoring the formation and progression of individual unwinding forks, we observe that both the frequency of initiation and rate of unwinding are highly dependent on RecQ concentration. We establish that unwinding forks can initiate internally by melting into dsDNA and can proceed in both directions. The findings suggest that initiation requires a RecQ dimer, and continued unwinding involves the repeated cooperative action of multiple monomers at the DNA unwinding fork. We propose a distinctive model wherein RecQ melts dsDNA internally to initiate unwinding, and subsequently unwinds DNA as a dynamic assembly of proteins cooperating at the fork.