Location of a P1 plasmid replication inhibitor determinant within the initiator gene

Muraiso, Kanae; Mukhopadhyay, Gauranga; Chattoraj, Dhruba K.

doi:10.1128/jb.172.8.4441-4447.1990

Cited by 12 publications

(10 citation statements)

References 24 publications

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“…However, overproduction of initiator proteins does not necessarily lead to an increase in copy number. The initiator proteins of plasmids P1, F, R6K, and Rtsl have been shown to directly exert both positive and negative effects on replication initiation (11,14,23,28,32). Although the mechanism of the direct inhibitory action of excess initiator protein is not yet fully understood, recent findings support the hypothesis that two different forms of the initiator protein may be responsible and that proteolytic processing may be involved (17,24).…”

supporting

confidence: 64%

The amount of RepR protein determines the copy number of plasmid pIP501 in Bacillus subtilis

Brantl¹,

Behnke²

1992

J Bacteriol

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To prove the hypothesis that the amount of RepR protein is the rate-limiting factor for replication of plasmid pIP501 in Bacillus subtilis, the repR gene was placed under control of the inducible promoter pspac. The plasmid copy number of the pIP501 derivative pRS9 could be deliberately adjusted between approximately 1 and 50 to 100 molecules per cell by varying the concentration of the inducer isopropyl-beta-D-thiogalactopyranoside. Construction of a repR-lacZ fusion proved that the increase in copy number was due to a proportional increase in the amount of RepR protein.

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supporting

confidence: 64%

The amount of RepR protein determines the copy number of plasmid pIP501 in Bacillus subtilis

Brantl¹,

Behnke²

1992

J Bacteriol

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“…Mutating the translation start codon of repA reduces RepA production drastically but not the synthesis of a replication inhibitor (9). Also excess of purified RepA does not cause replication inhibition in vitro (36,38).…”

Section: Discussionmentioning

confidence: 98%

“…Two host proteins, DnaA and HU (4,5), and P1 RepA (6) are essential for initiation of P1 replication, and excess iterons or excess RepA inhibit P1 replication (7)(8)(9). These characteristics of plasmid replication were found to be also true for the opening reaction, validating the use of the opening assay to study initiation control.…”

mentioning

confidence: 85%

Requirements for and Regulation of Origin Opening of Plasmid P1

Park

Mukhopadhyay

Chattoraj

1998

Journal of Biological Chemistry

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Origin opening is essential for the initiation of DNA replication in the theta mode and requires binding of initiator proteins. Using reactivity to KMnO 4 in vivo as an assay, we find that, like initiation, origin opening of the Escherichia coli plasmid P1 requires the host initiators DnaA and HU and the plasmid-encoded initiator RepA. The ability to detect opening at the P1ori in vivo allowed us to study this activity at various copy numbers in chimeric replicons. The opening was prevented when the P1ori was cloned in high copy vectors or when excess RepA binding sites (iterons) were provided in trans. However, when RepA supply was also increased, the opening was efficient. A further increase in RepA prevented opening. Replication of an incoming P1 under these conditions correlated with opening. These results demonstrate that initiation is possible even at abnormally high origin concentrations and that oversupply of RepA, relative to iterons, can prevent replication by blocking origin opening. It appears that plasmid overreplication can be prevented either by limiting RepA or by accumulating RepA at a rate higher than that of the origin.Initiation of DNA replication has been staged into discrete steps primarily from the work in vitro on plasmids carrying the Escherichia coli origin, oriC. Binding of initiators first opens the origin, and the opening provides the stage for the DnaCmediated loading of the DnaB helicase (1). In the absence of the DnaB-DnaC complex, a stable open state of the origin could be obtained in vitro. Using a dnaC(ts) mutant host at the nonpermissive temperature, a stable open structure could also be obtained in vivo as assayed by reactivity to KMnO 4 (2). Reactivity was lost rapidly when the culture was shifted down to permissive temperature. It appears that once DnaB is loaded, the subsequent steps of priming and elongation can proceed rapidly and are unlikely to be the steps for controlling initiation frequency.We have examined whether origin opening of plasmid P1 can be used to follow regulation of initiation in vivo. Plasmid P1 belongs to a family of plasmids characterized by the presence of short repeating sequences, called iterons (3). The iterons are binding sites for the plasmid-encoded initiator, RepA. Iterons cover about half of the minimal origin (ori) of P1 and also constitute the control locus, incA (see Fig. 1). The incA locus can be deleted, and such plasmids are maintained at an 8-fold higher copy number. The locus therefore plays only a negative regulatory role in plasmid replication. The origin iterons (called incC; see Fig. 1) are essential for initiation and are believed to be important for the control of copy number as well. The incC locus also includes the promoter of the initiator gene. Consequently, RepA binding to incC results in efficient repression of the RepA promoter. Autoregulated initiator synthesis is generally a conserved feature of iteron-carrying plasmids, implying that maintenance of initiator concentration is critical to the copy number control process...

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“…1 (Table 2). The observed inhibitory effect of excess RepA in wild-type cells was expected (37), although the mechanism of this inhibition is not understood at present. In the absence of any of the three HSPs, small amounts of RepA could not support the replication of the P1 plasmid, but increasing the RepA concentration 7 or 40-fold allowed replication to nearly the extent obtained in wild-type cells.…”

Section: -531mentioning

confidence: 92%

Heat shock proteins DnaJ, DnaK, and GrpE stimulate P1 plasmid replication by promoting initiator binding to the origin

Sozhamannan

Chattoraj

1993

J Bacteriol

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Binding of the P1-encoded protein RepA to the origin of P1 plasmid replication is essential for initiation of DNA replication and for autoregulatory repression of the repA promoter. Previous studies have shown defects in both initiation and repression in hosts lacking heat shock proteins DnaJ, DnaK, and GrpE and have suggested that these proteins play a role in the RepA-DNA binding required for initiation and repression. In this study, using in vivo dimethyl sulfate footprinting, we have confirmed the roles of the three heat shock proteins in promoting RepA binding to the origin. The defects in both activities could be suppressed by increasing the concentration of wild-type RepA over the physiological level. We also isolated RepA mutants that were effective initiators and repressors without requiring the heat shock proteins. These data suggest that the heat shock proteins facilitate both repression and initiation by promoting only the DNA-binding activity of RepA. In a similar plasmid, F, initiator mutants that confer heat shock protein independence for replication were also found, but they were defective for repression. We propose that the initiator binding involved in repression and the initiator binding involved in initiation are similar in P1 but different in F.

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Location of a P1 plasmid replication inhibitor determinant within the initiator gene

Cited by 12 publications

References 24 publications

The amount of RepR protein determines the copy number of plasmid pIP501 in Bacillus subtilis

The amount of RepR protein determines the copy number of plasmid pIP501 in Bacillus subtilis

Requirements for and Regulation of Origin Opening of Plasmid P1

Heat shock proteins DnaJ, DnaK, and GrpE stimulate P1 plasmid replication by promoting initiator binding to the origin

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