Mini-P1 plasmid replication: The autoregulation-sequestration paradox

Chattoraj, Dhruba K.; Mason, Rebecca J.; Wickner, Sue

doi:10.1016/0092-8674(88)90468-0

Cited by 93 publications

(81 citation statements)

References 25 publications

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“…In plasmid P1, RepA-mediated looping of the replication origin to a second set of iterons is thought to provide an additional level of copy number regulation beyond intermolecular coupling (8,41). In plasmid R6K, the arrangement of the single and partial iterons of the a and P origins of replication, respectively, and the multiple iterons of the -y origin bear striking resemblance to the RK2 onV region, except that the predicted loops are larger in R6K (35).…”

Section: Methodsmentioning

confidence: 99%

Structure, expression, and regulation of the kilC operon of promiscuous IncP alpha plasmids

Larsen

Figurski

1994

J Bacteriol

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The kil-kor regulon was first identified on the broad-host-range IncPa plasmid RK2 by the presence of multiple kil loci (kiL4, kilB, kiC, and recently kiLE) that are lethal to Escherichia coli host cells in the absence of regulation by kor functions in various combinations. Whereas nine iterons of oriV. Iteron 10 is identical to the "orphan" iteron 1, and both have identical 6-bp flanking sequences that make them likely to be strong binding sites for the TrfA replication initiator protein. The locations and relative orientation of orphan iterons 10 and 1 raise the possibility that these iterons promote the formation of a DNA loop via protein-protein interactions by bound TrfA and lead us to propose that they demarcate the functional origin of replication. This analysis of the kilC region and our previous studies on the other kil loci of RK2 have revealed that the region between oriV and the korABF operon in wild-type IncPa plasmids is saturated by the kiC, kHlE, and kiL4 loci arranged in four kor-regulated operons encoding a total of 12 genes.The self-transmissible plasmids of incompatibility group P (IncPa and IncP,) are known for their conjugal and replicative promiscuity among diverse bacteria (10,40,42,55). Studies with the 60-kb IncPa plasmids RK2 (24) and RP4 (10) have revealed a unique regulatory network known as the kil-kor regulon (15,16,29). The eight operons of the kil-kor regulon are controlled by various combinations of transcriptional repressors encoded by korA, korB, and korC (15,29,55). The expression of several of the operons is further influenced by other regulatory functions encoded by korE, korFI, korFII, trbA, and kfrA (42,55,65). The functions of some of the proteins encoded by the kil-kor regulon are known (42). The trfA operon specifies the essential replication initiator function (TrfA), which binds to multiple copies of a 17-bp sequence (iteron) in the origin of replication (oriV), and a singlestranded DNA-binding protein (SSB). The korA operon includes the regulatory genes korA, korB, korFI and korFII, and

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Section: Methodsmentioning

confidence: 99%

Structure, expression, and regulation of the kilC operon of promiscuous IncP alpha plasmids

Larsen

Figurski

1994

J Bacteriol

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“…Repression of the repA promoter by RepA supplied in trans was used as a measure of RepA-iteron interactions in vivo (12). The assay system was as described except that the RepA source plasmid, pMVG02, was deleted for the incA iterons 9 ϩ 8Ј present in the previously used plasmid pRJM362.…”

Section: Methodsmentioning

confidence: 99%

“…Autoregulation normally counteracts protein reduction by titration: as titration reduces the free initiator concentration, autorepression is proportionally relieved and compensates for the reduction (11). To keep titration from counteracting autorepression, it was proposed that the titrated initiators pair with promoter-bound initiators and thereby help maintain the repression (12). This mechanism, now called handcuffing (13), is common among transcriptional repressors that loop DNA, where the titrated repressors, instead of relieving autorepression, actually increase it (14)(15)(16)(17).…”

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confidence: 99%

Multiple homeostatic mechanisms in the control of P1 plasmid replication

Das

Valjavec-Gratian

Basuray

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Many organisms control initiation of DNA replication by limiting supply or activity of initiator proteins. In plasmids, such as P1, initiators are limited primarily by transcription and dimerization. However, the relevance of initiator limitation to plasmid copy number control has appeared doubtful, because initiator oversupply increases the copy number only marginally. Copy number control instead has been attributed to initiator-mediated plasmid pairing (''handcuffing''), because initiator mutations to handcuffing deficiency elevates the copy number significantly. Here, we present genetic evidence of a role for initiator limitation in plasmid copy number control by showing that autorepression-defective initiator mutants also can elevate the plasmid copy number. We further show, by quantitative modeling, that initiator dimerization is a homeostatic mechanism that dampens active monomer increase when the protein is oversupplied. This finding implies that oversupplied initiator proteins are largely dimeric, partly accounting for their limited ability to increase copy number. A combination of autorepression, dimerization, and handcuffing appears to account fully for control of P1 plasmid copy number.autorepression ͉ DNA replication control ͉ homeostatic control ͉ plasmid copy number control

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“…DNA molecules were prepared for microscopy by following essentially the method of Davis et al and others [28,291. DNA samples (concentrations =lo pg/ml in Tns/ EDTA buffer) were mixed with cytochrome c (0.01%) and 50% formamide and then spread on water.…”

Section: Electron Microscopymentioning

confidence: 99%

Characterisation and mode of in vitro replication of pea chloroplast OriA sequences

Reddy

Choudhury

Kumar

et al. 1994

European Journal of Biochemistry

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A partially purified replicative system of pea chloroplast that replicates recombinant DNAs containing pea chloroplast origin sequences has been characterised. Polymerisation by this system is very fast and insensitive to chain terminators like dideoxynucleotides, arabinosylcytosine 5'-triphosphate, etc. Both strands of template DNA are synthesized and single-stranded DNA templates undergo more than one round of replication.When sequences of either of the two chloroplast origins of replication (OriA or OriB) are used as templates, the replicative intermediates are found to have sigma structures. Electron microscopic analysis of the sigma structures restricted with various enzymes reveals that the initiation site of in vitro replication maps near the displacement-loop regions where replication initiates also in vivo. Although the observed replication initiation in the OriA recombinant template is chloroplast-DNAspecific, the mode of replication is different from that observed in vivo with intact ctDNA. However, when the template DNA contains both the OriA and OriB sequences, the in vitro replication proceeds in the theta mode, the mode of replication usually observed in vivo.The complex process of DNA replication has been defined for only a few biological systems such as plasmids, bacteriophages, bacteria and viruses [l-61. So far, little is known about the DNA replication processes in plant systems except for few reports in the field of plant organelles [7-111. Chloroplast DNA (ctDNA) of higher plants seems to be an attractive system for investigation of the molecular details of replication. The chloroplast genomic size of about 150 kb is large but not too large for recombinant manipulations and the genome exists in multiple copies. Understanding the molecular biology of ctDNA replication would not only reveal the detailed characteristics of this system [12-141 but also help solve problems related to stable transformation of the chloroplast organelle. Thus, development of an in vitro replication system derived from higher plant chloroplasts is important both in helping to define DNA metabolism in general as well as advancing the technology of transgenic plant production [15-181. Replicative intermediates of ctDNA from pea leaves have been well characterised using electron microscopic techniques [19]. In pea and maize ctDNA, it was shown that replication begins by forming two displacement loops (Dloops) which expand towards each other to form a theta structure. This small Cairns' structure then proceeds bi-directionally until termination takes place. Meeker et al. [20] have mapped the two replication origins or D-loops by electron Correspondence to S . K.

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Mini-P1 plasmid replication: The autoregulation-sequestration paradox

Cited by 93 publications

References 25 publications

Structure, expression, and regulation of the kilC operon of promiscuous IncP alpha plasmids

Structure, expression, and regulation of the kilC operon of promiscuous IncP alpha plasmids

Multiple homeostatic mechanisms in the control of P1 plasmid replication

Characterisation and mode of in vitro replication of pea chloroplast OriA sequences

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