A protein that binds to the P1 origin core and the oriC 13mer region in a methylation-specific fashion is the product of the host seqA gene.

Brendler, Therese; Abeles, Ann L.; Austin, Stuart

doi:10.1002/j.1460-2075.1995.tb00080.x

Cited by 109 publications

(90 citation statements)

References 27 publications

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“…Examples of studies of the possible role of SeqA in the regulation of replication initiated from origins different from oriC include relatively preliminary observations only, namely the demonstration of the binding of this protein to bacteriophage P1 ori (Brendler et al, 1995), and the findings that the copy number of plasmids derived from bacteriophage l is decreased in a seqA mutant host (S omiń ska et al, 2001) and that the DseqA mutation can suppress incompatibility between l plasmids and certain dnaA(ts) mutants (S omiń ska et al, 2003b;Glinkowska et al, 2003). Here, we investigated the role of SeqA in the regulation of l DNA replication in more detail.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2007

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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 l plasmid, is decreased, and that the activity of the l p R promoter (whose function is required for stimulation of oril) is lower than that in the wild-type host. Here, SeqA-mediated regulation of l phage and plasmid replicons was investigated in more detail. No significant influence of SeqA on oril-dependent DNA replication in vitro was observed, indicating that a direct regulation of l DNA replication by this protein is unlikely. On the other hand, density-shift experiments, in which the fate of labelled l DNA was monitored after phage infection of host cells, strongly suggested the early appearance of s replication intermediates and preferential rolling-circle replication of phage DNA in seqA mutants. The directionality of l plasmid replication in such mutants was, however, only slightly affected. The stability of the heritable l replication complex was decreased in the seqA mutant relative to the wild-type host, but a stable fraction of the l 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 l DNA replication, the efficiency of l plasmid replication in amino acid-starved relA seqA mutants was measured. Under these conditions, seqA dysfunction resulted in impairment of l plasmid replication. These results indicate that unlike oriC, SeqA modulates l DNA replication indirectly, most probably by influencing the stability of the l replication complex and the transcriptional activation of oril. INTRODUCTIONReplication of genetic material is a fundamental process that occurs in all living organisms (Kornberg & Baker, 1992). To ensure survival, this process must be precisely regulated, and this regulation usually occurs at the stage of initiation. In the model prokaryotic organism Escherichia coli, initiation of chromosome replication is controlled in the cell cycle (Messer, 2002). The dnaA gene product is a replication initiator protein. It binds to the oriC region and positively regulates DNA replication initiation. However, in most biological systems, which require precise and versatile regulation to ensure the ability to respond to various growth conditions, there are both positive and negative regulators. The SeqA protein appears to be one of key factors involved in the negative control of oriCinitiated replication (Lu et al., 1994;von Freiesleben et al., 1994;Slater et al., 1995;Boye et al., 1996); namely, it is an inhibitor of the onset of chromosome replication in vivo (Slater et al., 1995;Boye et al., 1996).It has been demonstrated that in vivo SeqA limits DnaA activity in replication from oriC (von Freiesleben et al., 1994). Nevertheless, it has been proposed that the main role of SeqA is seque...

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Section: Discussionmentioning

confidence: 99%

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

et al. 2007

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“…DNA fragments were isolated by polyacrylamide electrophoresis, purified, and labelled by filling in the ends with the appropriate [␣-32 P]-dNTP and Klenow enzyme as previously described (Brendler et al, 1995). The DNA sequence of all synthetic origin constructs was confirmed by sequencing (Radnedge et al, 1996).…”

Section: Plasmid Dna Proceduresmentioning

confidence: 99%

The iteron bases and spacers of the P1 replication origin contain information that specifies the formation of a complex structure involved in initiation

Brendler

Abeles

Reaves

et al. 1997

Molecular Microbiology

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SummaryThe origin of replication of the P1 plasmid contains five direct, imperfect repeats (iterons) of a 19 bp sequence that binds the P1-encoded RepA initiator protein. RepA binding to these iterons triggers origin initiation and represses transcription from the repA promoter that is nested within the iterons. The origin iterons were replaced with ligated oligonucleotides that insert five perfect 19 bp repeats with identical spacer sequences. This eliminates the natural variation in the iteron and spacer sequences and removes the repA promoter. The reconstructed origin is functional, showing that the repA promoter is not essential for origin function. The method used to make the reconstructed origin allows substitution of identical iterons with altered sequence or spacer length. Single changes of conserved iteron bases gave reduced or non-existent origin activity, as did an increase in spacer length. Like the wild type, most of these mutant arrays retain avid primary binding activity for the RepA protein. However, although the wild-type arrays readily form a mature complex in which all iterons are saturated, the most replication-defective mutants were completely unable to do this, even at very high RepA concentrations. It appears that iteron spacing and contacts involving at least three of the conserved iteron bases play an important role in the assembly of the mature structure in which all sites are occupied. A model is presented in which an allosteric interaction between the DNA site and protein is needed for the saturated, mature complex required for initiation.

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“…This process functions by specific inactivation of newly replicated origins by the protein SeqA (Slater et al, 1995). Newly synthesized DNA in E. coli is hemimethylated, methylated (by Dam methylase) on the old strand and unmethylated on the new strand, and SeqA binds preferentially to hemimethylated DNA (Brendler et al, 1995;Slater et al, 1995). In V. cholerae, similar control mechanisms may operate, since this organism also harbours seqA and dam genes (Hiraga et al, 2000;Løbner-Olesen et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

Replication patterns and organization of replication forks in Vibrio cholerae

2011

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We have investigated the replication patterns of the two chromosomes of the bacterium Vibrio cholerae grown in four different media. By combining flow cytometry and quantitative real-time PCR with computer simulations, we show that in rich media, V. cholerae cells grow with overlapping replication cycles of both the large chromosome (ChrI) and the small chromosome (ChrII). In Luria-Bertani (LB) medium, initiation occurs at four copies of the ChrI origin and two copies of the ChrII origin. Replication of ChrII was found to occur at the end of the ChrI replication period in all four growth conditions. Novel cell-sorting experiments with marker frequency analysis support these conclusions. Incubation with protein synthesis inhibitors indicated that the potential for initiation of replication of ChrII was present at the same time as that of ChrI, but was actively delayed until much of ChrI was replicated. Investigations of the localization of SeqA bound to new DNA at replication forks indicated that the forks were co-localized in pairs when cells grew without overlapping replication cycles and in higher-order structures during more rapid growth. The increased degree of fork organization during rapid growth may be a means by which correct segregation of daughter molecules is facilitated.

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A protein that binds to the P1 origin core and the oriC 13mer region in a methylation-specific fashion is the product of the host seqA gene.

Cited by 109 publications

References 27 publications

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

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

The iteron bases and spacers of the P1 replication origin contain information that specifies the formation of a complex structure involved in initiation

Replication patterns and organization of replication forks in Vibrio cholerae

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