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Stoichiometry and Architecture of Active DNA Replication Machinery in Escherichia coli

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Science  23 Apr 2010:
Vol. 328, Issue 5977, pp. 498-501
DOI: 10.1126/science.1185757

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Forking Replisomes

Replisomes are multiprotein machines that replicate DNA. Significant insight into how they work comes from in vitro studies, but how replisomes are organized in living cells has remained unclear. Reyes-Lamothe et al. (p. 498) have watched the replisome in living Escherichia coli cells using single-molecule fluorescence spectroscopy with millisecond time resolution. Cells expressing fluorescent derivatives of 10 different replisome components revealed both the stoichiometry and spatial distribution of the components at active replication forks in Escherichia coli. A similar technique could be used to study other molecular machines as they function.

Abstract

The multiprotein replisome complex that replicates DNA has been extensively characterized in vitro, but its composition and architecture in vivo is unknown. Using millisecond single-molecule fluorescence microscopy in living cells expressing fluorescent derivatives of replisome components, we have examined replisome stoichiometry and architecture. Active Escherichia coli replisomes contain three molecules of the replicative polymerase, rather than the historically accepted two. These are associated with three molecules of τ, a clamp loader component that trimerizes polymerase. Only two of the three sliding clamps are always associated with the core replisome. Single-strand binding protein has a broader spatial distribution than the core components, with 5 to 11 tetramers per replisome. This in vivo technique could provide single-molecule insight into other molecular machines.

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