Replication of the Escherichia coli chromosome is initiated at a unique site, oriC. Concurrent initiation occurs at all oriC sites present in a cell once, and only once, per cell cycle. A mechanism to ensure cyclic initiation events was found operating through the chromosomal site, datA, a 1-kb segment located at 94.7 min on the genetic map that titrates exceptionally large amounts of the bacterial initiator protein, DnaA. A strain lacking datA grew normally but exhibited an asynchronous initiation phenotype as a result of extra initiation events. This mutant phenotype was suppressed by DnaA-titrating plasmids. Furthermore, mutations in a 9-bp DnaA-binding sequence (the DnaA box) in datA were enough to induce the mutant phenotype. Thus, datA is a novel chromosomal element that appears to adjust a balance between free and bound DnaA for a single initiation event at a fixed time in the bacterial cell cycle. Titration of DnaA to newly duplicated datA during oriC sequestration, which is mediated by hemimethylated GATC sequences in oriC and the SeqA protein, would contribute to prevention of reinitiations when oriC is desequestered.

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There is a DNA sequence which has unusually high affinity for the DnaA protein of Escherichia coli between the glyV and amiB-mutL operons at 94.7 min on the genetic map. Affinity of DnaA protein for DNA was measured in vivo as the activity of beta-galactosidase produced from the lacZ gene on a plasmid fused to the 5'-terminal portion of the mioC gene, which is under the control of the DnaA protein. The chromosomal DNA segment between the two operons, carried on a compatible plasmid, derepressed the beta-galactosidase activity by titrating DnaA protein. Derepression occurred on the chromosomal dnaA gene as well, since it is autoregulated. Hence, as measured by immunoassays, one plasmid molecule carrying the DnaA-binding region titrated 370 dnaA molecules, which is a value eightfold higher than that for a plasmid containing the oriC region. We estimate that about 60% of the total cellular DnaA molecules are bound to this site. Four DnaA-binding sequences (DnaA boxes) and a DnaA-regulated promoter directing transcription of two small genes were present within a 250 bp stretch in this region but additional long DNA regions, including the fifth DnaA box located about 650 bp downstream, were required for maximum binding. A role for the DnaA-binding site in controlling DnaA-protein concentration in the cell cycle is discussed.

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Escherichia coli rnh mutants were isolated using localized mutagenesis and selective measurements of RNase H activity in mutagenized cell extracts with [3H]poly(rC) X poly(dG) as substrate. RNase H activity in extracts of one mutant, ON152 (rnh-91), was undetectable (less than 0.05% of that of wild-type cells). This mutant formed small colonies at 43 degrees C. At this temperature, accumulation of nascent fragments was more prominent in the rnh-91 X polA4113 double mutant than in the polA4113 mutant; however, no accumulation was found in the rnh single mutant at 43 degrees C. Unlike the 1-3 nucleotide primer RNA found on nascent fragments of polA4113 cells, primers from the rnh-91 X polA4113 cells ranged from one to about ten bases. These results suggest that the 5' leads to 3' exonuclease activity of DNA polymerase I plays a major role in removal of primer RNA and that RNase H functions in an auxiliary role, excising the 5'-portion of longer primers. The rnh mutant supports replication of ColE1-type plasmids. A possible mechanism of replication of such plasmids in rnh mutants and a role of RNase H in the initiation of chromosomal replication are discussed.