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Modulation of plasmid and phage DNA replication by Escherichia coli SeqA protein

Sylwia Baraska¹

Alicja Wgrzyn²

Grzegorz Wgrzyn¹

¹ Department of Molecular Biology, University of Gdask, 80-822 Gdask, Poland
² Laboratory of Molecular Biology (affiliated with University of Gdask), Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 80-822 Gdask, Poland

Correspondence
Grzegorz Wgrzyn
wegrzyn{at}biotech.univ.gda.pl

SeqA protein, a main negative regulator of the replication initiation of the Escherichia coli chromosome, also has several other functions which are still poorly understood. It was demonstrated previously that in seqA mutants the copy number of another replicon, the plasmid, is decreased, and that the activity of the p_R promoter (whose function is required for stimulation of ori) is lower than that in the wild-type host. Here, SeqA-mediated regulation of phage and plasmid replicons was investigated in more detail. No significant influence of SeqA on ori-dependent DNA replication in vitro was observed, indicating that a direct regulation of DNA replication by this protein is unlikely. On the other hand, density-shift experiments, in which the fate of labelled DNA was monitored after phage infection of host cells, strongly suggested the early appearance of replication intermediates and preferential rolling-circle replication of phage DNA in seqA mutants. The directionality of plasmid replication in such mutants was, however, only slightly affected. The stability of the heritable replication complex was decreased in the seqA mutant relative to the wild-type host, but a stable fraction of the O protein was easily detectable, indicating that such a heritable complex can function in the mutant. To investigate the influence of seqA gene function on heritable complex- and transcription-dependent DNA replication, the efficiency of plasmid replication in amino acid-starved relA seqA mutants was measured. Under these conditions, seqA dysfunction resulted in impairment of plasmid replication. These results indicate that unlike oriC, SeqA modulates DNA replication indirectly, most probably by influencing the stability of the replication complex and the transcriptional activation of ori.