Specificity determinants in interaction of the initiator (Rep) proteins with the origins in the plasmids ColE2-P9 and ColE3-CA38 identified by chimera analysis.
The ColE2-P9 rep protein specifically binds to the orgin and initiates DNA synthesis. Interaction of the Rep protein with the origins of plasmids ColE2-P9 and ColE-3-CA38 (one of the close relatives of ColE2-P9) is plasmid-specific. By using chimeric rep genes and chimeric origins we showed that the two region, A and B, in the C-terminal regions of the Rep proteins and the two sites alpha and beta, in the origins are important for the determination of specificity. When each of the A/alpha and B/beta pairs is from the same plasmids, the plasmid replication is efficient. On the other hand, if only the A/alpha pair is from the same plasmids, the plasmid replication is inefficient. For the region A, the plasmid-specificity is mainly determined by the presence or absence of a nine-amino acid sequence. For the region B, the specificity is probably determined by several amino acids. The region B, contains a segment of amino acid sequence which shows significant homology with the DNA recognition helices of various DNA binding proteins. At the site alpha, the single additional base-pair in the ColE3-CA38 origin can be either A/T or T/A. At the site beta, however, the single additional base-pair in the ColE2-P9 origin must be G/C. Among other possibilities we propose that the region A is a linker connecting the two domains in the Rep protein involved in DNA-binding and that the region B is a part of the sequence-specific DNA-binding domain.
Shinohara
M
M
Department of Biology, Faculty of Science, Osaka University, Japan.
Itoh
T
T
D000595:Amino Acid Sequence; D003086:Bacteriocin Plasmids / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication / Q000235:genetics; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D018741:Replication Origin / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D004265:DNA Helicases