Abstract
Preferential binding of SeqA protein to hemimethylated oriC, the origin of Escherichia coli chromosomal replication, delays methylation by Dam methylase. Because the SeqA-oriC interaction appears to be essential in timing of chromosomal replication initiation, the biochemical functions of SeqA protein and Dam methylase at the 13-mer L, M, and R region containing 4 GATC sequences at the left end of oriC were examined. We found that SeqA protein preferentially bound hemimethylated 13-mers but not fully nor unmethylated 13-mers. Regardless of strand methylation, the binding of SeqA protein to the hemimethylated GATC sequence of 13-mer L was followed by additional binding to other hemimethylated GATC sequences of 13-mer M and R. On the other hand, Dam methylase did not discriminate binding of 13-mers in different methylation patterns and was not specific to GATC sequences. The binding specificity and higher affinity of SeqA protein over Dam methylase to the hemimethylated 13-mers along with the reported cellular abundance of this protein explains the dominant action of SeqA protein over Dam methylase to the newly replicated oriC for the sequestration of chromosomal replication. Furthermore, SeqA protein bound to hemimethylated 13-mers was not dissociated by Dam methylase, and most SeqA protein spontaneously dissociated 10 min after binding. Also, SeqA protein delayed the in vitro methylation of hemimethylated 13-mers by Dam methylase. These in vitro results suggest that the intrinsic binding instability of SeqA protein results in release of sequestrated hemimethylated oriC.
Highlights
We found that SeqA protein preferentially bound hemimethylated 13-mers but not fully nor unmethylated 13-mers
During replication of the E. coli chromosome, newly synthesized strands exist as unmethylated species until methylated by Dam methylase [21]
Because oriC contains 11 repeats of GATC sequence [18], the complexity of binding kinetic analysis and localization of SeqA protein to hemimethylated oriC were reduced by using the 13-mer region of oriC containing only 4 GATC sequences (Fig. 1)
Summary
Reagent—Sources were as follows: [␥-32P]ATP (5000 Ci/mmol), Amersham Pharmacia Biotech; poly(dI-dC) and Sephadex-G50, Amersham Pharmacia Biotech; long ranger polyacrylamide, FMC Corp. Gel-shift Assay—To obtain differently methylated duplex 13-mers (Fig. 1), synthesized oligonucleotides containing 13-mer regions were mixed with appropriate combinations, 32P-labeled with T4 polynucleotide kinase and [␥-32P]ATP, heated, and annealed by slow cooling. 20 l of binding mixture contained 1 g of poly(dIdC) and 1.5 fmol of 32P-labeled hemimethylated 13-mers, unless indicated, in binding buffer (10 mM Tris-HCl (pH 7.6), 50 mM KCl, 1 mM EDTA, 1 mM DTT, and 10% glycerol). The indicated amounts of proteins were added to 20 l of the binding mixture described under “Gel-shift Assay” and incubated for 20 min at 32 °C. 500 fmol of indicated 13-mers was added to 20 l of Dam assay mixture (0.1 M Tris-HCl (pH 8.0), 10 mM EDTA, 2.5 mM DTT, and 1.6 M S-adenosyl-[methyl-3H]-methionine. Filters were washed with 0.4 M ammonium bicarbonate, further washed with cold ethanol, dried, and quantitated using a liquid scintillation counter
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