Simultaneous single-molecule measurements of phage T7 replisome composition and function reveal the mechanism of polymerase exchange.

ABSTRACT: A complete understanding of the molecular mechanisms underlying the functioning of large, multiprotein complexes requires experimental tools capable of simultaneously visualizing molecular architecture and enzymatic activity in real time. We developed a novel single-molecule assay that combines the flow-stretching of individual DNA molecules to measure the activity of the DNA-replication machinery with the visualization of fluorescently labeled DNA polymerases at the replication fork. By correlating polymerase stoichiometry with DNA synthesis of T7 bacteriophage replisomes, we are able to quantitatively describe the mechanism of polymerase exchange. We find that even at relatively modest polymerase concentration (∼2 nM), soluble polymerases are recruited to an actively synthesizing repliso

SUBMITTER: Loparo JJ 

PROVIDER: S-EPMC3048139 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature