Abstract
A DNA replication system was developed that could generate rolling-circle DNA molecules in vitro in amounts that permitted kinetic analyses of the movement of the replication forks. Two artificial primer-template DNA substrates were used to study DNA synthesis catalyzed by the DNA polymerase III holoenzyme in the presence of either the preprimosomal proteins (the primosomal proteins minus the DNA G primase) and the Escherichia coli single-stranded DNA binding protein or the DNA B helicase alone. Helicase activities have recently been demonstrated to be associated with the primosome, a mobile multiprotein priming apparatus that requires seven E. coli proteins (replication factor Y (protein n'), proteins n and n'', and the products of the dnaB, dnaC, dnaG, and dnaT genes) for assembly, and with the DNA B protein. Consistent with a rolling-circle mechanism in which a helicase activity permitted extensive (-) strand DNA synthesis on a (+) single-stranded, circular DNA template, the major DNA products formed were multigenome-length, single-stranded, linear molecules. The replication forks assembled with either the preprimosome or the DNA B helicase moved at the same rate (approximately 730 nucleotides/s) at 30 degrees C and possessed apparent processivities in the range of 50,000-150,000 nucleotides. The single-stranded DNA binding protein was not required to maintain this high rate of movement in the case of leading strand DNA synthesis catalyzed by the DNA polymerase III holoenzyme and the DNA B helicase.
Highlights
A DNA replication system was developed that could generaterolling-circle DNA molecules invitro in amounts that permitted kinetic analyses of the movement of the replication forks
The DNA B protein [10, 11] was originally identifiedas a component of the primosome ( l a ), a mobile multiprotein apparatus involved in the initiation of 4 x 1 7 4 SS(c)+replicative form (RF)DNA replication [13, 14].While the requirementfor the DNA B protein along withsix otherproteins(the DNA C protein,the DNA G
Primase, factorY, proteini, protein n, and protein n”) to effect efficient primosome assembly has been established, the components that remain thereafter ptoerform the actual priming and strand-displacing functions remaeiqnuivocal. Studies from this laboratory on the formation of rollingcircle molecules during the 4 x 1 7 4 SS(c) + RF DNA replication reaction in vitro [9] suggested that this type of DNA synthesis mightprove an informativeprobe for investigating how the Pol I11 HE functions in combination with a stranddisplacing helicase activity at the replication fork
Summary
The DNA B protein [10, 11] was originally identifiedas a component of the primosome ( l a ) , a mobile multiprotein apparatus involved in the initiation of 4 x 1 7 4 SS(c)+replicative form (RF)DNA replication [13, 14].While the requirementfor the DNA B protein along withsix otherproteins(the DNA C protein,the DNA G primase, factorY (protein n’), proteini, protein n, and protein n”) to effect efficient primosome assembly has been established, the components that remain thereafter ptoerform the actual priming and strand-displacing functions remaeiqnuivocal Studies from this laboratory on the formation of rollingcircle molecules during the 4 x 1 7 4 SS(c) + RF DNA replication reaction in vitro [9] suggested that this type of DNA synthesis mightprove an informativeprobe for investigating how the Pol I11 HE functions in combination with a stranddisplacing helicase activity at the replication fork. The generationof rollingcircle DNA products in this system idsescribed, and evidence is presented that threplication forks assembled move at high rates (equivalent to those measured in uiuo) and are highly processive
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
