Primer RNA synthesis by plasmid-specified Rep protein for initiation of ColE2 DNA replication.

Takechi, Shinji; Matsui, Hideji; Itoh, Tateo

doi:10.1002/j.1460-2075.1995.tb00196.x

Cited by 33 publications

(25 citation statements)

References 36 publications

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“…The protein is necessary for replication. The ColE2 Rep to which it is similar is a plasmid-specific primase which binds to double-stranded DNA and synthesizes a unique RNA primer (43,44). ColE2-type plasmids need host-encoded proteins, e.g., DNA polymerase I for the initial DNA synthesis and DNA polymerase III for completing the synthesis (13,14,42,46).…”

Section: Discussionmentioning

confidence: 99%

Protein-DNA interactions in the ori region of the Mycobacterium fortuitum plasmid pAL5000

Stolt¹,

Stoker²

1996

J Bacteriol

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Plasmid pAL5000 from Mycobacterium fortuitum encodes two proteins necessary for replication: RepA (307 amino acid residues) and RepB (119 residues). A single RNA species encoding these proteins was characterized, and its 5 end was defined. The proteins were expressed as maltose-binding protein fusions in Escherichia coli. The RepB protein was shown in vitro to bind specifically to a previously defined 435-bp region of pAL5000 containing the origin of replication (ori). The precise RepB binding sites were defined by DNase I footprinting experiments. RepB binds to two motifs in the ori region: a high-affinity site within its own promoter region, implying autoregulation of its expression, and a low-affinity site further upstream, presumably the origin of replication itself. The binding to the latter motif seems to occur on one DNA strand only. The high-affinity binding site contains several palindromic sequences. Gel retardation assays were performed with the different binding sites as templates, and the binding constant to each site was estimated from protein titrations. This is the first molecular dissection of mycobacterial DNA-binding proteins and their interactions with their targets.The mechanisms governing plasmid replication and maintenance in mycobacteria are little understood. Plasmids have been described in several species, e.g., Mycobacterium fortuitum, Mycobacterium scrofulaceum, Mycobacterium chelonae, and Mycobacterium avium (4,8,17,18,23,29). No plasmids have been found in the pathogen Mycobacterium tuberculosis, but it is possible to transform M. tuberculosis with plasmids from related mycobacteria, showing that the lack of plasmids is not due to an intrinsic inability to support their replication.The replicon most used in genetic manipulation of mycobacteria is the 4,837-bp M. fortuitum plasmid pAL5000 (19) (see Fig. 1) (GenBank accession number M23557). This replicon is the basis of several shuttle plasmids and cosmids used to manipulate DNA in a wide variety of mycobacteria, including the fast-growing Mycobacterium smegmatis and the slow growers Mycobacterium bovis BCG and M. tuberculosis (9,31,36,41). We have previously shown that the origin of replication (ori) of this plasmid lies within a 435-bp segment and comprises a region with a high incidence of repeated sequences (39). In addition, it has been shown that two open reading frames (ORFs) are necessary for a functional replicon (22,39). The products of these ORFs, RepA and RepB, will act in trans to activate the ori if this region is present on another plasmid (39).The ori region is also able to confer incompatibility to otherwise unrelated replicons. Whether the same sequence motifs within the region are responsible for the inc and ori functions or whether inc is physically separated from ori as in plasmid P1 (1, 5, 7) is at present unclear.The RepA protein (307 amino acid [aa]) has a high similarity to replication proteins from ColE2-related plasmids (12, 39). RepB is a small protein (119 aa) which has recently been shown to have similar...

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Section: Discussionmentioning

confidence: 99%

Protein-DNA interactions in the ori region of the Mycobacterium fortuitum plasmid pAL5000

Stolt¹,

Stoker²

1996

J Bacteriol

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“…The ColE2 Rep protein is unique among other initiator proteins in that it is also a plasmid-specific primase. It synthesizes a threenucleotide primer RNA which has a unique structure of 5Ј ppApGpA (33). Host DNA polymerase I specifically uses the primer RNA to start DNA synthesis and then form a D-loop structure, into which various replication proteins of E. coli, such as DnaB helicase, DnaG primase, and the DNA polymerase III holoenzyme, are introduced to continue replication of ColE2 DNA.…”

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Specific Interaction between the Initiator Protein (Rep) and Origin of Plasmid ColE2-P9

2007

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The replication initiator protein (Rep) of plasmid ColE2-P9 (ColE2) is multifunctional. We are interested in how Rep binds to the origin (Ori) to perform various functions. We used the wild type and variants of Rep to study the Rep-Ori interaction by both in vitro and in vivo approaches, including biochemical analyses of protein-DNA interactions and an in vivo replication assay. We identified three regions (I, II, and III) of Rep, located in the C-terminal half, and three corresponding binding sites (I, II, and III) in Ori which are important for Rep-Ori interaction. We showed that region I, containing a putative helix-turn-helix motif, is necessary and sufficient for specific Ori recognition, interacting with site I of the origin DNA from the major groove. Region II interacts with site II of the origin DNA, from the adjacent minor groove in the left half of Ori, and region III interacts with site III, next to the template sequence for primer synthesis, which is one and one-half turn apart from site I on the opposite surface of the origin DNA. A putative linker region located between the two DNA binding domains (regions II and III) was identified, which might provide Rep an extended conformation suitable for binding to the two separate sites in Ori. Based on the results presented in this paper, we propose a model for Rep-Ori interaction in which Rep binds to Ori as a monomer.

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“…Initiation of the plasmid replication requires host DNA polymerase I (16,26) and a plasmid-encoded replication initiator (Rep) protein that uniquely possesses an origin-specific primase activity among bacterial plasmids (15,35), and replication proceeds in a unidirectional manner (13,28). The Rep protein specifically binds to the replication origin (15,33) and synthesizes a short RNA molecule of 5Ј-ppApG pA-3Ј at a specific position in the origin as a primer for initiation of DNA synthesis by DNA polymerase I (28,29). The 32-bp minimal ColE2 origin may be divided into three functional subregions, as proposed by in vivo analyses using an exhaustive mutant set of single-base-pair substitutions (33).…”

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Distinct Functions of the Two Specificity Determinants in Replication Initiation of Plasmids ColE2-P9 and ColE3-CA38

2007

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The plasmid ColE2-P9 Rep protein specifically binds to the cognate replication origin to initiate DNA replication. The replicons of the plasmids ColE2-P9 and ColE3-CA38 are closely related, although the actions of the Rep proteins on the origins are specific to the plasmids. The previous chimera analysis identified two regions, regions A and B, in the Rep proteins and two sites, ␣ and ␤, in the origins as specificity determinants and showed that when each component of the region A-site ␣ pair and the region B-site ␤ pair is derived from the same plasmid, plasmid DNA replication is efficient. It is also indicated that the replication specificity is mainly determined by region A and site ␣. By using an electrophoretic mobility shift assay, we demonstrated that region B and site ␤ play a critical role for stable Rep protein-origin binding and, furthermore, that 284-Thr in this region of the ColE2 Rep protein and the corresponding 293-Trp of the ColE3 Rep protein mainly determine the Rep-origin binding specificity. On the other hand, region A and site ␣ were involved in the efficient unwinding of several nucleotide residues around site ␣, although they were not involved in the stable binding of the Rep protein to the origin. Finally, we discussed how the action of the Rep protein on the origin involving these specificity determinants leads to the plasmid-specific replication initiation.In all organisms, DNA replication is a key event for inheritance of genetic information. Initiation of DNA replication requires interaction of the initiator protein with the specific DNA region called the replication origin and the consequent localized melting of duplex DNA, which provides a singlestranded template for establishment of replication machinery. In initiation of chromosomal DNA replication in Escherichia coli, several molecules of the initiator protein (DnaA) tightly bind to the 9-mer repeated sequences called the DnaA boxes in the chromosomal replication origin (oriC), and then the DnaB helicase is loaded onto the unwound 13-mer AT-rich region located to one side of oriC, followed by establishment of the replication machinery containing DnaG and DNA polymerase III holoenzyme (17).The plasmid ColE2-P9 (ColE2) is a circular duplex DNA molecule of about 7 kb (10) and is kept at 10 to 15 copies per chromosome (2, 12). Initiation of the plasmid replication requires host DNA polymerase I (16, 26) and a plasmid-encoded replication initiator (Rep) protein that uniquely possesses an origin-specific primase activity among bacterial plasmids (15,35), and replication proceeds in a unidirectional manner (13, 28). The Rep protein specifically binds to the replication origin (15, 33) and synthesizes a short RNA molecule of 5Ј-ppApG pA-3Ј at a specific position in the origin as a primer for initiation of DNA synthesis by DNA polymerase I (28, 29). The 32-bp minimal ColE2 origin may be divided into three functional subregions, as proposed by in vivo analyses using an exhaustive mutant set of single-base-pair substitutions (33). One of th...

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Primer RNA synthesis by plasmid-specified Rep protein for initiation of ColE2 DNA replication.

Cited by 33 publications

References 36 publications

Protein-DNA interactions in the ori region of the Mycobacterium fortuitum plasmid pAL5000

Protein-DNA interactions in the ori region of the Mycobacterium fortuitum plasmid pAL5000

Specific Interaction between the Initiator Protein (Rep) and Origin of Plasmid ColE2-P9

Distinct Functions of the Two Specificity Determinants in Replication Initiation of Plasmids ColE2-P9 and ColE3-CA38

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