Single-molecule studies of fork dynamics in Escherichia coli DNA replication.
We present single-molecule studies of the Escherichia coli replication machinery. We visualize individual E. coli DNA polymerase III (Pol III) holoenzymes engaging in primer extension and leading-strand synthesis. When coupled to the replicative helicase DnaB, Pol III mediates leading-strand synthesis with a processivity of 10.5 kilobases (kb), eight-fold higher than that by Pol III alone. Addition of the primase DnaG causes a three-fold reduction in the processivity of leading-strand synthesis, an effect dependent upon the DnaB-DnaG protein-protein interaction rather than primase activity. A single-molecule analysis of the replication kinetics with varying DnaG concentrations indicates that a cooperative binding of two or three DnaG monomers to DnaB halts synthesis. Modulation of DnaB helicase activity through the interaction with DnaG suggests a mechanism that prevents leading-strand synthesis from outpacing lagging-strand synthesis during slow primer synthesis on the lagging strand.
Tanner
Nathan A
NA
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Avenue, Boston, Massachusetts 02115, USA.
Hamdan
Samir M
SM
Jergic
Slobodan
S
Loscha
Karin V
KV
Schaeffer
Patrick M
PM
Dixon
Nicholas E
NE
van Oijen
Antoine M
AM
D004258:DNA Polymerase III / Q000378:metabolism*; D019915:DNA Primase; D004261:DNA Replication / Q000502:physiology*; D004269:DNA, Bacterial / Q000378:metabolism*; D053488:DnaB Helicases / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D011485:Protein Binding
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D019915:DNA Primase; D004258:DNA Polymerase III; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; C121791:dnaG protein, E coli; C506730:dnaB protein, E coli; D053488:DnaB Helicases