Interaction of the initiator protein of an IncB plasmid with its origin of DNA replication.
The replication initiator protein RepA of the IncB plasmid pMU720 was purified and used in DNase I protection assays in vitro. RepA protected a 68-bp region of the origin of replication of pMU720. This region, which lies immediately downstream of the DnaA box, contains four copies of the sequence motif 5'AANCNGCAA3'. Mutational analyses identified this sequence as the binding site specifically recognized by RepA (the RepA box). Binding of RepA to the RepA boxes was ordered and sequential, with the box closest to the DnaA binding site (box 1) occupied first and the most distant boxes (boxes 3 and 4) occupied last. However, only boxes 1, 2, and 4 were essential for origin activity, with box 3 playing a lesser role. Changing the spacing between box 1 and the other three boxes affected binding of RepA in vitro and origin activity in vivo, indicating that the RepA molecules bound to ori(B) interact with one another.
Betteridge
T
T
Department of Microbiology and Immunology, The University of Melbourne, Melbourne, Victoria 3010, Australia.
Yang
J
J
Pittard
A J
AJ
Praszkier
J
J
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D018983:DNA Footprinting; D004265:DNA Helicases; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018741:Replication Origin; D015534:Trans-Activators
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D029968:Escherichia coli Proteins; D011506:Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D003850:Deoxyribonuclease I; D004265:DNA Helicases