Replication enhancer-independent mutation increases the co-operativity with which an initiator protein binds its origin.
The plus-strand replication origin of bacteriophage fl 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 (beta-gamma) 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.
Greenstein
D
D
Rockefeller University, New York, NY 10021.
Horiuchi
K
K
D001483:Base Sequence; D003001:Cloning, Molecular; D003090:Coliphages / Q000235:genetics*; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D004926:Escherichia coli / Q000235:genetics*; D005814:Genes, Viral; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D014764:Viral Proteins / Q000378:metabolism*
C024013:DNA replication complex protein, Bacteriophage lambda; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D014764:Viral Proteins