Dna Unwinding Dynamics of a Processive DNA Polymerase

Abstract

During DNA replication mechanical unwinding of the DNA helix is required for the advance of the replication machinery. Unlike many DNA polymerases the bacteriophage Phi29 DNA polymerase presents a processive ‘helicase-like’ activity and is able to couple DNA replication and unwinding within the same polypeptide. Using Optical Tweezers we have developed a single molecule mechanical assay to elucidate the physical mechanism of DNA unwinding by the Phi29 DNA polymerase as the protein replicates processively the DNA. A DNA hairpin is hold between an optical trap and a mobile surface. As a single polymerase works on the hairpin its replication and unwinding activities can be measured in real time (by measuring the change in extension in the DNA polymer), revealing the fluctuations of their rates in response to the DNA sequence and force applied in the direction of unwinding. The sequence and force sensitivities of the unwinding reaction of the wild type and an unwinding-deficient polymerase mutant indicate that the Phi29 DNA polymerase presents an active unwinding mechanism that may substantially differ from the unwinding mechanism used by specialized nucleic acid helicases.