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

Kathryn M. Stephens, Roger McMacken

Research output: Contribution to journalArticlepeer-review

14 Scopus citations

Abstract

We have used a set of bacteriophage λ and Escherichia coli replication proteins to establish rolling circle DNA replication in vitro to permit characterization of the functional properties of λ replication forks. We demonstrate that the λ replication fork assembly synthesizes leading strand DNA chains at a physiological rate of 650-750 nucleotides/s at 30 °C. This rate is identical to the fork movement rate we obtained using a minimal protein system, composed solely of E. coli DnaB helicase and DNA polymerase III holoenzyme. Our data are consistent with the conclusion that these two key bacterial replication proteins constitute the basic functional unit of a λ replication fork. A comparison of rolling circle DNA replication in the minimal and A replication systems indicated that DNA synthesis proceeded for more extensive periods in the λ system and produced longer DNA chains, which averaged nearly 200 kilobases in length. The higher potency of the A replication system is believed to result from its capacity to mediate efficient reloading of DnaB helicase onto rolling circle replication products, thereby permitting reinitiation of DNA chain elongation following spontaneous termination events. E. coli single-stranded DNA-binding protein and primase individually stimulated rolling circle DNA replication, but they apparently act indirectly by blocking accumulation of inhibitory free single- stranded DNA product. Finally, in the course of this work, we discovered that E. coli DNA polymerase III holoenzyme is itself capable of carrying out significant strand displacement DNA synthesis at about 50 nucleotides/s when it is supplemented with E. coli single-stranded DNA-binding protein.

Original languageEnglish (US)
Pages (from-to)28800-28813
Number of pages14
JournalJournal of Biological Chemistry
Volume272
Issue number45
DOIs
StatePublished - Nov 7 1997
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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