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

Kojo Mensa-Wilmot; Ruth Seaby; Christine Alfano; Marc S Wold; Bruce Gomes; Roger McMacken

doi:10.1016/S0021-9258(19)81691-5

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

Journal article

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

Kojo Mensa-Wilmot, Ruth Seaby, Christine Alfano, Marc S Wold, Bruce Gomes and Roger McMacken

The Journal of biological chemistry, Vol.264(5), pp.2853-2861

1989

DOI: 10.1016/S0021-9258(19)81691-5

PMID: 2536726

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https://doi.org/10.1016/S0021-9258(19)81691-5View

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Abstract

We have established an in vitro system, composed of highly purified bacteriophage lambda and Escherichia coli proteins, that specifically replicates supercoiled templates bearing the lambda replication origin (ori lambda). The complete system is composed of three groups of proteins: the virus-encoded initiator proteins (the lambda 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 ori lambda and proceeds unidirectionally rightwards through theta-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 lambda 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 lambda replication system.

Molecular Genetics

Biological and medical sciences

Fundamental and applied biological sciences. Psychology

Molecular and cellular biology

Replication

Details

Title: Subtitle: Reconstitution of a nine-protein system that initiates bacteriophage λ DNA replication
Creators: Kojo Mensa-Wilmot - Johns Hopkins University
Ruth Seaby - Johns Hopkins University
Christine Alfano - Johns Hopkins University
Marc S Wold - Johns Hopkins University
Bruce Gomes - Johns Hopkins University
Roger McMacken - Johns Hopkins University
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.264(5), pp.2853-2861
DOI: 10.1016/S0021-9258(19)81691-5
PMID: 2536726
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Publisher: American Society for Biochemistry and Molecular Biology
Language: English
Date published: 1989
Academic Unit: Radiation Oncology; Biochemistry and Molecular Biology
Record Identifier: 9984293086502771

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