Replication enhancer-independent mutation increases the co-operativity with which an initiator protein binds its origin

David Greenstein, Kensuke Horiuchi

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

Abstract

The plus-strand replication origin of bacteriophage f1 has a bipartite structure consisting of a required core origin region and an adjacent A + T-rich enhancer sequence that potentiates replication approximately 100-fold. The core origin binds the initiator protein (gpII) and the enhancer binds the Escherichia coli integration host factor (IHF). gpII binds the core origin in two steps, forming a binding intermediate (complex I) and a functional complex for nicking (complex II). We have used a double-label gel binding assay to determine the stoichiometry of the gpII-origin interaction. The results indicate that complex I contains two gpII molecules per origin, and complex II contains four gpII molecules per origin. Enhancer-independent mutations in gpII allow wild-type levels of replication in the absence of either the enhancer or IHF. We have examined the binding of an enhancer-independent gpII mutant (mp1) protein to the replication origin. The mp1 mutation in gpII (Met40 → Ile) increases the co-operativity with which the protein binds to form complex II. In addition, the mutant gpII forms both complexes with a DNA fragment containing only two (β-γ) of the three repeats from the core origin sequence, while the wild-type protein forms only complex I with this fragment. We discuss how a mutation that increases the co-operativity of binding of an initiator protein might stimulate DNA replication.

Original languageEnglish (US)
Pages (from-to)91-101
Number of pages11
JournalJournal of Molecular Biology
Volume211
Issue number1
DOIs
StatePublished - Jan 5 1990

Bibliographical note

Funding Information:
We thank Peter Ilodel. David Russell and Norton Zinder for helpful discussions and caritical reading of the manuscript, and Howard Nash fbr valuable suggestions. Amy Roth provided excellent technical assistance. This work was supported by grants from bhr Sat,ional Sciencae Foundation and the Nabional Institutes of Healt,h. I).(:. was supported by t,raining grant 4107233 from the National Institutes of Health.

Copyright:
Copyright 2014 Elsevier B.V., All rights reserved.

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