Stoichiometry and architecture of active DNA replication machinery in Escherichia coli.
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 tau, 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.
Reyes-Lamothe
Rodrigo
R
Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.
Sherratt
David J
DJ
Leake
Mark C
MC
D004258:DNA Polymerase III / Q000032:analysis* / Q000378:metabolism; D004261:DNA Replication; D004269:DNA, Bacterial / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000032:analysis / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000032:analysis* / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000032:analysis* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009097:Multienzyme Complexes / Q000032:analysis* / Q000378:metabolism; D009690:Nucleic Acid Conformation; D000099272:Replisomes
D004258:DNA Polymerase III; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D000099272:Replisomes; C476430:SSB protein, E coli; C101139:beta subunit, DNA polymerase III