Plus-origin mapping of single-stranded DNA plasmid pE194 and nick site homologies with other plasmids

Sozhamannan, Shanmuga; Dabert, Patrick; Moretto, V; Ehrlich, S. Dusko; Gruss, Alexandra

doi:10.1128/jb.172.8.4543-4548.1990

Cited by 25 publications

(15 citation statements)

References 39 publications

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“…The pLS1 family of plasmids includes 10 replicons (9, 10), to which recently described plasmid pCI411 (5) should be added. The Rep nick site has been unequivocally determined for pLS1 (6,20) and genetically shown for pE194 (30). With these two exceptions, little is known about the leading-strand initiation and control region of the other members of the family, except for the information derived from sequence homologies.…”

Section: Resultsmentioning

confidence: 99%

In vitro recognition of the replication origin of pLS1 and of plasmids of the pLS1 family by the RepB initiator protein

1995

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Rolling-circle replication of plasmid pLS1 is initiated by the plasmid-encoded RepB protein, which has nicking-closing (site-specific DNA strand transferase) enzymatic activity. The leading-strand origin of pLS1 contains two regions, (i) the RepB-binding site, constituted by three directly repeated sequences (iterons or the bind region), and (ii) the sequence where RepB introduces the nick to initiate replication (the nic region). A series of plasmids, belonging to the pLS1 family, show features similar to those of pLS1 and have DNA sequences homologous to the pLS1 nic region. In addition, they all share homologies at the level of their Rep proteins. However, the bind regions of these plasmids are, in general, not conserved. We tested the substrate specificity of purified RepB of pLS1. The RepB protein has a temperature-dependent nicking-closing action on supercoiled pLS1, as well as on recombinant plasmid DNAs harboring the pLS1 nic region. The DNA strand transferase activity of pLS1-encoded RepB was also assayed on two plasmids of the pLS1 family, namely, pE194 and pFX2. DNAs from both plasmids were relaxed by RepB, provided they had a proper degree of supercoiling; i.e., it was necessary to modulate the supercoiling of pE194 DNA to achieve RepB-mediated DNA relaxation. Single-stranded oligonucleotides containing the nic regions of various plasmids belonging to the pLS1 family, including those of pE194 and pFX2, were substrates for RepB. In vitro, the RepB protein does not need to bind to the iterons for its nicking-closing activity.Many multicopy plasmids isolated from gram-positive and -negative bacteria replicate by an asymmetric rolling-circle (RC) mechanism (reviewed in references 10, 13, and 23). Characterization of various staphylococcal replicons has led to the proposal that plasmids replicating by the RC mode are constructed as interchangeable genetic modules (26). Only one of these modules is essential for plasmid replication, the leading-strand initiation and control region (10). On the basis of homologies in the DNA sequence and in the genetic organization of the leading-strand initiation and control region, four families have been defined, and their prototypes are pT181, pSN2, pC194/pUB110, and pMV158 (10, 13, 23). The latter plasmid has been studied in some detail for its derivative pLS1, which lacks the mob gene involved in conjugative mobilization (19,25). The leading-strand initiation and control region includes the plasmid double-strand origin (dso) and the gene encoding the initiator of replication (Rep) protein. The dso can be physically and functionally divided into two regions, (i) bind, where the Rep protein binds, and (ii) nic, which includes the Rep-mediated nick site used to initiate replication (11). The two regions can be contiguous (as in pT181) or separated by dozens of bases (as in pLS1). The nic region contains one or two stem-loop structures which have been mapped by determining the sensitivity of plasmids pLS1 and pT181 to nuclease S1 in vitro and in vivo (21, 27). The DNA seq...

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Section: Resultsmentioning

confidence: 99%

In vitro recognition of the replication origin of pLS1 and of plasmids of the pLS1 family by the RepB initiator protein

1995

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“…In a unit-length circular molecule, initiation and termination of ssDNA synthesis should occur at the same site within the plus-strand origin of replication. If two plus-strand origins of replication are present in the same molecule, as is the case in molecules containing tandem viral genome repeats, plus-strand synthesis may initiate at one origin and terminate at the other (19)(20)(21)(22). Single-strand replication from a molecule containing tandem genome repeats derived from different viral strains is predicted to generate unit-length progeny genomes of a single predominant genotype.…”

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confidence: 99%

Replicational release of geminivirus genomes from tandemly repeated copies: evidence for rolling-circle replication of a plant viral DNA.

Stenger

Revington

Stevenson

et al. 1991

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

304

223

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Agrobacterium-mediated inoculation of Nicotiana benthamiana plants with Ti plasmids containing tandem genome repeats derived from different strains of the geminivirus beet curly top virus (BCTV) resulted in the production of unit-length recombinant progeny genomes in systemically infected plants. When two putative plus-strand orgins of replication were present in constructs used as inocula, a replicational escape mechanism was favored that resulted in progeny genomes of a single predominant genotype. The genotype was dependent upon the arrangement of repeated parental genomes in the inocula. Sequencing across thejunction between parental BCTV strains in the recombinant progeny allowed mapping of the plus-strand origin of replication to a 20-base-pair sequence within the conserved hairpin found in all geminivirus genomes. In contrast, when inocula contained tandemly repeated BCTV genome sequences but only a single conserved hairpin, a number of different progeny genotypes were simultaneously replicated in infected plants, a result expected if unit-length viral genomes were generated by random intramolecular recombination events. These results and other considerations indicate that geminivirus DNA replication occurs by a rollingcircle mechanism.

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“…The SS DNA is then converted to the double-stranded (DS) form utilizing the single-strand origin (SSO) (Baas and Jansz, 1988;Birch and Khan, 1992;Dempsey et al, 1995;Gruss et al, 1987). Previous studies have shown that the origin of replication contains sequences necessary for both the initiation and termination of RC replication (Dotto et al, 1984;Gros et al, 1987;Murray et al, 1989;Sozhamannan et al, 1990;Iordanescu and Projan, 1988;Zhao and Khan, 1996). While the sequences required for initiation and termination overlap, a larger region is required for the initiation step (Gros et al, 1987;Zhao and Khan, 1996).…”

Section: Introductionmentioning

confidence: 99%

Sequence requirements for the termination of rolling‐circle replication of plasmid pT181

Zhao

Khan

1997

Molecular Microbiology

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SummaryMost small multicopy plasmids of Gram-positive bacteria and many in Gram-negative bacteria replicate by a rolling-circle (RC) mechanism. The replication initiator proteins encoded by the RC plasmids and single-stranded bacteriophages of Escherichia coli have origin-specific nicking-closing activities that are required for the initiation and termination of RC replication. We have investigated the sequence requirements for termination of RC replication of plasmid pT181. The initiator nick site is located in the loop of a hairpin region (IRII) within the pT181 origin of replication. By mutational analysis, we have found that several nucleotides within the stem of IRII which are critical for the initiation activity are dispensable for termination of replication. We also demonstrate that nucleotides in the right arm of IRII, but not the left arm, are absolutely required for termination of RC replication. We have also identified specific nucleotides in IRII that are critical for its termination activity. The sequence of the right arm of the hairpin must be located downstream of the initiator nick site for termination, suggesting that termination requires a specific orientation of the initiator protein at the origin.

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Plus-origin mapping of single-stranded DNA plasmid pE194 and nick site homologies with other plasmids

Cited by 25 publications

References 39 publications

In vitro recognition of the replication origin of pLS1 and of plasmids of the pLS1 family by the RepB initiator protein

In vitro recognition of the replication origin of pLS1 and of plasmids of the pLS1 family by the RepB initiator protein

Replicational release of geminivirus genomes from tandemly repeated copies: evidence for rolling-circle replication of a plant viral DNA.

Sequence requirements for the termination of rolling‐circle replication of plasmid pT181

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