No abstract

The P1 plasmid replication origin P1oriR is controlled by methylation of four GATC adenine methylation sites within heptamer repeats. A comparable (13mer) region is present in the host origin, oriC. The two origins show comparable responses to methylation; negative control by recognition of hemimethylated DNA (sequestration) and a positive requirement for methylation for efficient function. We have isolated a host protein that recognizes the P1 origin region only when it is isolated from a strain proficient for adenine methylation. The substantially purified 22 kDa protein also binds to the 13mer region of oriC in a methylation‐specific fashion. It proved to be the product of the seqA gene that acts in the negative control of oriC by sequestration. We conclude that the role of the SeqA protein in sequestration is to recognize the methylation state of P1oriR and oriC by direct DNA binding. Using synthetic substrates we show that SeqA binds exclusively to the hemimethylated forms of these origins forms that are the immediate products of replication in a methylation‐proficient strain. We also show that the protein can recognize sequences with multiple GATC sites, irrespective of the surrounding sequence. The basis for origin specificity is primarily the persistence of hemimethylated forms that are over‐represented in the natural. DNA preparations relative to controls.

Replication functions of a bacteriophage P1 miniplasmid are carried on a 1.2-kilobase pair (kb) segment that can be subdivided into a 245-base pair (bp) replication origin and a 959-bp region that encodes a protein required for replication (RepA). The origin region contains five 19-bp direct repeats. By using primer extension and gene-fusion assays, we mapped the promoter of the repA gene within the repeated sequences and showed that the promoter is repressed byRepA. Regulation of RepA synthesis is apparently achieved by the binding of RepA to the repeat sequences. This regulation might be a key step in the replication-control circuit, as we found that overproduction of RepA (from a foreign promoter) inhibits replication. Thus, in addition to being an autoregulated activator of replication, the protein also can have a negative regulatory role. RepA. We also show that the repA gene is autoregulated and that overproduction of the protein (from a foreign promoter) interferes with plasmid replication. Thus, RepA, although required for replication, also can act as a negative regulator. MATERIALS AND METHODSIsolation of Total Cellular RNA. A 5-ml culture of a recA56 derivative of MC4100 (6) carrying the plasmid pALA326 (see Fig. 4) was grown in L broth to OD590 = 0.5; harvested by centrifugation; resuspended in 0.3 ml of a solution containing 30 mM NaOAc (pH 4.8), 0.5% NaDodSO4, and 1 mM EDTA solution; and extracted with an equal volume of phenol at 60'C for 5 min (7). The aqueous layer was reextracted with phenol/CHCl3, 1:1 (vol/vol), and then was precipitated with ethanol. The pellet was dissolved in the NaOAc solution (as above) and reprecipitated with ethanol. The process was repeated once more, and the dry pellet was dissolved in 100 ,u of 10 mM Tris, pH 7.5/10 mM MgSO4; 50 ,ug of DNase I (RNase free; ref. 8) was then added. After incubation at 37°C for 20 min, the mixture was extracted with phenol, precipitated with ethanol, and dissolved in 100 Al of H20. A 40-Al portion of the RNA was hylbridized to 32P-labeled DNA primer in a solution containing 80% formamide, 1 mM EDTA, 0.4 M NaCl, and 40 mM 1,4-piperazinediethanesulfonic acid (pH 6.4) at 56°C overnight (9). After hybridization, the DNARNA hybrids were recovered by precipitation with ethanol. cDNA was synthesized in a reaction mixture containing 100 mM Tris-HCl ( Fig. 1) is cloned in pBR322, the resultant plasmid, pALA69, efficiently complements the replication defect of X-P1:5RrepA103, which is a mini-P1 plasmid with an amber mutation in the repA gene (5). pALA69 contains the coding sequences for the RepA protein and is devoid of flanking inc sequences except for 1.8 repeats at the incA end. We have shown previously that these residual sequences do not destabilize mini-P1 plasmids (ref. 4; Fig. 1). The activity, in trans, of RepA allowed us to map the cis-acting region of X-P1:SR that apparently includes the origin of replication (5). To determine the position of the origin, we shortened our previAbbreviations: kb, kilobase pair(s); bp, base pair(s...

The P1 plasmid replication origin requires the host DnaA protein for function. Two DnaA-binding boxes lie in tandem within the previously defined minimal origin, constituting its left boundary. Three more boxes lie 200 base pairs to the right of these, in the leader region for the P1 repA gene. We show that either set alone is active for origin function. One of the two origin boxes is relatively inactive. Constructs with just one of the five boxes are active for specific origin function as long as the box conforms exactly to the published consensus. This single consensus box is functional when placed either to the left or right of the core origin sequences. The flexibility shown by this system suggests that the boxes play a role different from those in the host oriC origin, where the number and position of boxes are critical.