Reconstitution of a nine-protein system that initiates bacteriophage λ DNA replication

K. Mensa-Wilmot, R. Seaby, C. Alfano, M. S. Wold, B. Gomes, R. McMacken

Research output: Contribution to journalArticle

Abstract

We have established an in vitro system, composed of highly purified bacteriophage λ and Escherichia coli proteins, that specifically replicates supercoiled templates bearing the λ replication origin (oriλ). The complete system is composed of three groups of proteins: the virus-encoded initiator proteins (the λ O and P proteins), the E. coli replication fork propagation machinery (single-stranded DNA-binding protein, dnaB helicase, dnaG primase, DNA polymerase III holoenzyme, and DNA gyrase), and two bacterial heat shock proteins (dnaJ and dnaK proteins). DNA replication in this system is initiated at or near oriλ and proceeds unidirectionally rightwards through θ-structure intermediates, ultimately yielding a pair of intertwined daughter circles as the final product. In striking contrast to the situation in vivo and in crude in vitro systems, initiation of γ DNA replication in the purified protein system does not require 'transcriptional activation' of the origin region by E. coli RNA polymerase. We conclude that E. coli primase generates the primers for all leading and lagging strand DNA chains synthesized in this reconstituted λ replication system.

Original languageEnglish (US)
Pages (from-to)2853-2861
Number of pages9
JournalJournal of Biological Chemistry
Volume264
Issue number5
StatePublished - Jan 1 1989

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Mensa-Wilmot, K., Seaby, R., Alfano, C., Wold, M. S., Gomes, B., & McMacken, R. (1989). Reconstitution of a nine-protein system that initiates bacteriophage λ DNA replication. Journal of Biological Chemistry, 264(5), 2853-2861.