Simultaneous single-molecule measurements of phage T7 replisome composition and function reveal the mechanism of polymerase exchange.
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 replisome, dramatically increasing local polymerase concentration. These excess polymerases remain passively associated with the replisome through electrostatic interactions with the T7 helicase for ∼50 s until a stochastic and transient dissociation of the synthesizing polymerase from the primer-template allows for a polymerase exchange event to occur.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Joseph J
JJ
Loparo
Arkadiusz W
AW
Kulczyk
Charles C
CC
Richardson
Antoine M
AM
van Oijen
D017123:Bacteriophage T7 / Q000201:enzymology* / Q000378:metabolism; D001671:Biochemistry / Q000379:methods*; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D007700:Kinetics; D009097:Multienzyme Complexes / Q000378:metabolism*; D050505:Mutant Proteins / Q000378:metabolism; D011485:Protein Binding; D012996:Solutions; D014764:Viral Proteins / Q000378:metabolism; D000099272:Replisomes
D004259:DNA-Directed DNA Polymerase; D009097:Multienzyme Complexes; D050505:Mutant Proteins; D012996:Solutions; D014764:Viral Proteins; D000099272:Replisomes
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