Open complex formation by DnaA initiation protein at the Escherichia coli chromosomal origin requires the 13-mers precisely spaced relative to the 9-mers.
The 245 bp chromosomal origin, oriC, of Escherichia coli contains two iterated motifs. Three 13-mers tandemly repeated at one end of the origin and four 9-mers in a nearby segment of oriC are highly conserved in enteric bacteria, as is the distance separating these two sequence clusters. Mutant origins were constructed with altered spacing of the 9-mers relative to the 13-mers. Loss or addition of even a single base drastically reduced replication, both in vivo and in vitro. Spacing mutant origins bound effectively to DnaA protein but failed to support efficient open complex formation. These results suggest that interaction with the 9-mers positions at least one subunit of DnaA to recognize directly the nearest 13-mer for DNA melting.
Department of Biochemistry, Merck Research Laboratories, Rahway, New Jersey 07065.
J
J
Hsu
D
D
Bramhill
C M
CM
Thompson
D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002876:Chromosomes, Bacterial / Q000378:metabolism*; D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011485:Protein Binding; D017384:Sequence Deletion
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria