Effects of purified SeqA protein on oriC-dependent DNA replication in vitro.
In vivo studies suggest that the Escherichia coli SeqA protein modulates replication initiation in two ways: by delaying initiation and by sequestering newly replicated origins from undergoing re-replication. As a first approach towards understanding the biochemical bases for these effects, we have examined the effects of purified SeqA protein on replication reactions performed in vitro on an oriC plasmid. Our results demonstrate that SeqA directly affects the biochemical events occurring at oriC. First, SeqA inhibits formation of the pre-priming complex. Secondly, SeqA can inhibit replication from an established pre-priming complex, without disrupting the complex. Thirdly, SeqA alters the dependence of the replication system on DnaA protein concentration, stimulating replication at low concentrations of DnaA. Our data suggest that SeqA participates in the assembly of initiation-competent complexes at oriC and, at a later stage, influences the behaviour of these complexes.
Wold
S
S
Department of Cell Biology, Institute for Cancer Research, Montebello, 0310 Oslo, Norway.
Boye
E
E
Slater
S
S
Kleckner
N
N
Skarstad
K
K
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000494:pharmacology* / Q000502:physiology; D004261:DNA Replication / Q000502:physiology*; D004269:DNA, Bacterial / Q000096:biosynthesis* / Q000235:genetics; D004268:DNA-Binding Proteins / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D010957:Plasmids / Q000235:genetics; D018741:Replication Origin / Q000502:physiology*; D014157:Transcription Factors
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D029968:Escherichia coli Proteins; C089746:SeqA protein, E coli; D014157:Transcription Factors