Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

Functional properties of replication fork assemblies established by the bacteriophage lambda O and P replication

K M Stephens1, R McMacken

  • 1Department of Biochemistry, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, Maryland 21205, USA.

The Journal of Biological Chemistry
|November 14, 1997
PubMed
Summary
This summary is machine-generated.

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Crystallization and preliminary crystallographic characterization of the origin-binding domain of the bacteriophage lambda O replication initiator.

Acta crystallographica. Section F, Structural biology and crystallization communications·2007
Same author

Functional defects of the DnaK756 mutant chaperone of Escherichia coli indicate distinct roles for amino- and carboxyl-terminal residues in substrate and co-chaperone interaction and interdomain communication.

The Journal of biological chemistry·1999
Same author

Recognition, targeting, and hydrolysis of the lambda O replication protein by the ClpP/ClpX protease.

The Journal of biological chemistry·1999
Same author

DnaJ dramatically stimulates ATP hydrolysis by DnaK: insight into targeting of Hsp70 proteins to polypeptide substrates.

Biochemistry·1999
Same author

I-PpoI and I-CreI homing site sequence degeneracy determined by random mutagenesis and sequential in vitro enrichment.

Journal of molecular biology·1998
Same author

Kinetic characterization of the ATPase cycle of the DnaK molecular chaperone.

Biochemistry·1998

Bacteriophage lambda and E. coli proteins enable rolling circle DNA replication in vitro. This system efficiently synthesizes long DNA chains, revealing key bacterial replication proteins as the fundamental unit of replication forks.

Area of Science:

  • Molecular Biology
  • Virology
  • Genetics

Background:

  • Rolling circle DNA replication is a mechanism used by various viruses and plasmids.
  • Understanding replication fork dynamics is crucial for comprehending DNA replication fidelity and speed.

Purpose of the Study:

  • To characterize the functional properties of bacteriophage lambda replication forks in vitro.
  • To identify the core protein components essential for efficient DNA replication fork progression.

Main Methods:

  • Establishment of an in vitro rolling circle DNA replication system using bacteriophage lambda and Escherichia coli replication proteins.
  • Characterization of leading strand DNA synthesis rates and DNA chain lengths produced by different replication systems.
  • Analysis of the roles of E. coli DnaB helicase, DNA polymerase III holoenzyme, single-stranded DNA-binding protein, and primase.

Related Experiment Videos

Main Results:

  • The lambda replication fork assembly synthesizes leading strand DNA at a physiological rate (650-750 nucleotides/s).
  • A minimal system of E. coli DnaB helicase and DNA polymerase III holoenzyme replicates DNA at a similar rate, suggesting they form the basic functional unit.
  • The lambda system supports more extensive DNA synthesis, producing longer chains (average ~200 kb) due to efficient DnaB helicase reloading.
  • E. coli single-stranded DNA-binding protein and primase stimulate replication indirectly by preventing inhibitory single-stranded DNA accumulation.
  • E. coli DNA polymerase III holoenzyme performs strand displacement synthesis at ~50 nucleotides/s with single-stranded DNA-binding protein.

Conclusions:

  • E. coli DnaB helicase and DNA polymerase III holoenzyme are the fundamental components of a bacteriophage lambda replication fork.
  • The bacteriophage lambda replication system enhances rolling circle replication efficiency through effective DnaB helicase management.
  • E. coli single-stranded DNA-binding protein and primase play auxiliary roles in maintaining replication processivity.