Initiation of bacteriophage lambda DNA replication in vitro with purified lambda replication proteins

M S Wold; J B Mallory; J D Roberts; J H LeBowitz; R McMacken

doi:10.1073/pnas.79.20.6176

Back

Initiation of bacteriophage lambda DNA replication in vitro with purified lambda replication proteins

Journal article

Open access

Peer reviewed

Initiation of bacteriophage lambda DNA replication in vitro with purified lambda replication proteins

M S Wold, J B Mallory, J D Roberts, J H LeBowitz and R McMacken

Proceedings of the National Academy of Sciences - PNAS, Vol.79(20), pp.6176-6180

10/1982

DOI: 10.1073/pnas.79.20.6176

PMID: 6216478

Files and links (1)

url

https://doi.org/10.1073/pnas.79.20.6176View

Published (Version of record) Open Access

Abstract

We have developed a soluble enzyme system that replicates exogenously added plasmid DNA (lambda dv) bearing the replication origin of the bacteriophage lambda chromosome. The system contains pure phage lambda O and P replication proteins and a partially purified mixture of Escherichia coli replication proteins [the enzyme system of Fuller, R.S., Kaguni, J.M. & Kornberg, A. (1981) Proc. Natl. Acad. Sci. USA 78, 7370-7374). The features of lambda dv replication in this system closely resemble the known characteristics of phage lambda DNA replication in vivo. The system (i) depends completely on exogenously supplied DNA, (ii) specifically replicates supercoiled plasmid DNA that contains a lambda replication origin, (iii) depends on both the lambda O protein and the lambda P protein, (iv) depends on RNA polymerase, (v) depends on host replication proteins (e.g., primase, dnaB protein, and several others that function in the priming of DNA synthesis in E. coli) as judged by antibody inhibitions, and (vi) replicates as much as 32% of added lambda dv plasmid DNA through a single complete round to generate catenated daughter molecules. Furthermore, replication of lambda dv DNA in vitro requires DNA gyrase and an ATP-regenerating system. It is notable that addition of lambda O and P proteins to the mixture of E. coli replication proteins inhibits replication of plasmids bearing the origin of the E. coli chromosome. Exploitation of this enzyme system should allow a detailed investigation of the biochemical mechanisms involved in bacteriophage lambda DNA replication and its regulation.

DNA Replication

Templates, Genetic

Viral Proteins - genetics

DNA, Superhelical - genetics

Bacteriophage lambda - genetics

Cell-Free System

Details

Title: Subtitle: Initiation of bacteriophage lambda DNA replication in vitro with purified lambda replication proteins
Creators: M S Wold
J B Mallory
J D Roberts
J H LeBowitz
R McMacken
Resource Type: Journal article
Publication Details: Proceedings of the National Academy of Sciences - PNAS, Vol.79(20), pp.6176-6180
DOI: 10.1073/pnas.79.20.6176
PMID: 6216478
NLM abbreviation: Proc Natl Acad Sci U S A
ISSN: 0027-8424
eISSN: 1091-6490
Publisher: National Academy of Sciences; United States
Grant note: GM-24282 / NIGMS NIH HHS
Language: English
Date published: 10/1982
Academic Unit: Radiation Oncology; Biochemistry and Molecular Biology
Record Identifier: 9984024555902771

Metrics

33 Record Views

115 Times Cited - Web of Science