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

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