<i>In vitro</i>cleavage of double- and single-stranded DNA by plasmid RSF1010-encoded mobilization proteins

Scherzinger, Eberhard; Otto, Sabine; Dobrinski, Beate

doi:10.1093/nar/20.1.41

Cited by 97 publications

(140 citation statements)

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“…Relaxase proteins are also active in relaxosomes containing auxiliary DNA-binding proteins of host or plasmid origin. IncF, IncW, IncP, and IncQ systems offer well studied examples (12)(13)(14)(15)(16)(17)(18)(19)(20)(21). Among IncF plasmids factors known to stimulate nic cleavage include E. coli integration host factor and plasmid proteins TraM and TraY (22)(23)(24)(25)(26)(27).…”

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confidence: 99%

Extent of Single-stranded DNA Required for Efficient TraI Helicase Activity in Vitro

Csitkovits

Zechner

2003

Journal of Biological Chemistry

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The IncF plasmid protein TraI functions during bacterial conjugation as a site-and strand-specific DNA transesterase and a highly processive 5 to 3 DNA helicase. The N-terminal DNA transesterase domain of TraI localizes the protein to nic and cleaves this site within the plasmid transfer origin. In the cell the C-terminal DNA helicase domain of TraI is essential for driving the 5 to 3 unwinding of plasmid DNA from nic to provide the strand destined for transfer. In vitro, however, purified TraI protein cannot enter and unwind nicked plasmid DNA and instead requires a 5 tail of singlestranded DNA at the duplex junction. In this study we evaluate the extent of single-stranded DNA adjacent to the duplex that is required for efficient TraI-catalyzed DNA unwinding in vitro. A series of linear partial duplex DNA substrates containing a central stretch of singlestranded DNA of defined length was created and its structure verified. We found that substrates containing >27 nucleotides of single-stranded DNA 5 to the duplex were unwound efficiently by TraI, whereas substrates containing 20 or fewer nucleotides were not. These results imply that during conjugation localized unwinding of >20 nucleotides at nic is necessary to initiate unwinding of plasmid DNA strands.Helicases are ubiquitous enzymes involved in nucleic acid metabolism (for review, see Refs. 1-4). The TraI protein of IncF plasmids (first characterized as DNA helicase I of Escherichia coli (5)) is essential for the transmission of bacterial genes during conjugation (6, 7). In that process a copy of a conjugative plasmid is transferred unidirectionally in single-stranded form from one bacterial cell to another (for reviews, see Refs. 8 and 9). The TraI protein of IncF plasmids contributes to conjugative transfer in several ways. It is a component of a nucleoprotein complex, the relaxosome, which assembles with site specificity at the plasmid origin of transfer (oriT). Relaxosomes initiate the series of DNA-processing reactions that prepare plasmid DNA for transfer to a recipient cell. They are common to all self-transmissible and mobilizable plasmids in different degrees of complexity (for reviews, see Refs. 8, 10, and 11). The simplest systems employ a plasmid-encoded DNA transesterase, or relaxase, protein that acts on a specific phosphodiester bond, nic, in the transfer origin. Cleavage at this site provides a point of origin for the directed 5Ј to 3Ј transmission of the plasmid genome to a recipient cell. Relaxase proteins are also active in relaxosomes containing auxiliary DNA-binding proteins of host or plasmid origin. IncF, IncW, IncP, and IncQ systems offer well studied examples (12-21). Among IncF plasmids factors known to stimulate nic cleavage include E. coli integration host factor and plasmid proteins . In the case of IncW plasmid R388, TrwA protein performs this role (13,14). The IncF system and the functionally analogous IncW-IncN family of DNA-mobilizing systems are (thus far) unique in that they additionally specify a DNA helicase activity esse...

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confidence: 99%

Extent of Single-stranded DNA Required for Efficient TraI Helicase Activity in Vitro

Csitkovits

Zechner

2003

Journal of Biological Chemistry

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“…1. Plasmid RSF1010 contains its specific relaxase and oriT (Scherzinger et al, 1992). Mobilization of this plasmid by R6K and its derivatives was used to define the region carrying the relaxase of R6K and its accessory proteins.…”

Section: Mapping the Region Of R6k Involved In Conjugative Transfermentioning

confidence: 99%

“…published by Flashner et al (1996) can be interpreted as the proteins TaxA and TaxC (DDP1 and DDP2), but not TaxC alone, being able to cleave the nic site in vivo, inferring that both proteins will also be required for relaxation in vitro, as is also the case for other relaxases, such as TraI of RP4 (Pansegrau et al, 1990) or MobA of RSF1010 (Scherzinger et al, 1992) The insertion of the ⍀ cassette in plasmid pSU4746 introduces transcription and translation stop signals within taxA. In order to ascertain whether the mutation affected ᮊ 1997 Blackwell Science Ltd, Molecular Microbiology, 24, 1157-1168 Fig.…”

Section: Mapping the Region Of R6k Involved In Conjugative Transfermentioning

confidence: 99%

Genes involved in conjugative DNA processing of plasmid R6K

Núñez

Avila

Cruz

1997

Molecular Microbiology

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SummaryThe conjugative transfer region of the IncX plasmid R6K (TRA X ) was analysed by transposon mutagenesis and DNA sequencing. Tn5 tac1 insertional mutations localized TRA X to a 14.8 kb segment containing the ␣ origin of transfer (oriT␣), genes involved in conjugative DNA-processing (Dtr X ) and genes involved in pilus synthesis and assembly (Mpf X ). A second functional oriT, oriT ␤, was located at a distance of 5.3 kb from oriT␣ and was outside TRA X . Mpf X occupied a segment of 10 kb, as judged by the location of insertions conferring resistance to infection by the X pilusspecific phage X-2. At both sides of Mpf X there were insertions that were Tra ¹ but X-2 sensitive, suggesting that the mutations were in Dtr X . This region was sequenced and three genes were identified: taxA, taxB, and taxC. The overall organization was oriT␣-taxA-taxC-Mpf X -taxB. taxC coded for a oriT-relaxase that belongs to the VirD2 family. taxA coded for a protein of 181 amino acids that showed similarity to TraY of F-like plasmids and to the Arc-repressor superfamily. TaxB showed similarity to TraG-like proteins, a protein superfamily probably involved in coupling the relaxosome to the DNA-transport apparatus. TaxA and TaxC are required for oriT nicking in vivo. The nicking reaction was mistakenly assumed by Flashner et al. (1996) to represent a feature of the vegetative replication origins. However, insertions or deletions disrupting taxA and taxC affected conjugation but not replication of R6K. Conversely, protein , which is absolutely required for replication of R6K, was not required for conjugative transfer. In addition, protein DDP3, which is also assumed to have a role in replication, was found to be a positive modulator of bacterial conjugation. Taken together, these results rule out a direct and essential involvement of conjugation proteins in R6K vegetative replication, and also rule out the requirement of replication protein for conjugation.

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“…It has been reported that conjugative plasmids, which belong to different incompatibility groups from the group IncF of F and F-like plasmids, encode endonucleases which have other activities both in cleaving the singlestranded DNA at a specific site and in rejoining the cleaved products (Bhattacharjee & Meyer 1991;Scherzinger et al 1992;Pansegrau et al 1993). It has also been reported that the TraI protein encoded by plasmid F also has the cleaving and rejoining activities of the single-stranded DNA (Sherman & Matson 1994) which is similar to those encoded by the plasmids belonging to different incompatibility groups.…”

Section: Introductionmentioning

confidence: 99%

Roles of TraI protein with activities of cleaving and rejoining the single‐stranded DNA in both initiation and termination of conjugal DNA transfer

Fukuda

Ohtsubo

1997

Genes to Cells

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In vitrocleavage of double- and single-stranded DNA by plasmid RSF1010-encoded mobilization proteins

Cited by 97 publications

References 24 publications

Extent of Single-stranded DNA Required for Efficient TraI Helicase Activity in Vitro

Extent of Single-stranded DNA Required for Efficient TraI Helicase Activity in Vitro

Genes involved in conjugative DNA processing of plasmid R6K

Roles of TraI protein with activities of cleaving and rejoining the single‐stranded DNA in both initiation and termination of conjugal DNA transfer

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