Characterization of the reaction product of the oriT nicking reaction catalyzed by Escherichia coli DNA helicase I

Matson, Steven W.; Nelson, William; Morton, Brian

doi:10.1128/jb.175.9.2599-2606.1993

Cited by 53 publications

(62 citation statements)

References 30 publications

(29 reference statements)

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“…This reaction is also referred to as a relaxation reaction since the product of the reaction (after protein denaturation) is a relaxed plasmid (7). As a result of the transesterification, TraI is covalently bound to the 5Ј end of the nicked DNA strand that will ultimately be transferred into the recipient cell (27). TraI is also a processive 5Ј-to-3Ј DNA helicase (1,2,21).…”

Section: Resultsmentioning

confidence: 99%

The F-Plasmid TraI Protein Contains Three Functional Domains Required for Conjugative DNA Strand Transfer

Matson

Ragonese²

2005

J Bacteriol

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The F-plasmid-encoded TraI protein, also known as DNA helicase I, is a bifunctional protein required for conjugative DNA transfer. The enzyme catalyzes two distinct but functionally related reactions required for the DNA processing events associated with conjugation: the site-and strand-specific transesterification (relaxase) reaction that provides the nick required to initiate strand transfer and a processive 5-to-3 helicase reaction that provides the motive force for strand transfer. Previous studies have identified the relaxase domain, which encompasses the first ϳ310 amino acids of the protein. The helicase-associated motifs lie between amino acids 990 and 1450. The function of the region between amino acids 310 and 990 and the region from amino acid 1450 to the C-terminal end is unknown. A protein lacking the C-terminal 252 amino acids (TraI⌬252) was constructed and shown to have essentially wild-type levels of transesterase and helicase activity. In addition, the protein was capable of a functional interaction with other components of the minimal relaxosome. However, TraI⌬252 was not able to support conjugative DNA transfer in genetic complementation experiments. We conclude that TraI⌬252 lacks an essential C-terminal domain that is required for DNA transfer. We speculate this domain may be involved in essential protein-protein interactions with other components of the DNA transfer machinery.Conjugative DNA strand transfer is a highly conserved mechanism for unidirectional transfer of genetic information among bacteria of the same species, from one species to another and, in some instances, between kingdoms (for reviews, see references 15, 22, 25, 30, 37, and 44). Importantly, this is the mechanism used by most plasmids and conjugative transposons to facilitate their spread throughout a bacterial population, and the same underlying mechanism is used to transfer T-DNA from Agrobacterium tumefaciens to plant cells (23,45).The process of conjugative DNA transfer (CDT) is initiated with the formation of a stable mating pair between donor and recipient to establish the close cell-cell contact required for physical transfer of single-stranded DNA (ssDNA) from one cell to another. This is followed by the production of a site-and strand-specific nick at a locus called nic within the origin of transfer (oriT) or at the ends of T-DNA and the unwinding/ replication of the duplex DNA molecule to provide the ssDNA that enters the recipient. Within the recipient cell, host enzymes (primarily) convert the transferred ssDNA into doublestranded DNA that either circularizes to form a plasmid or is recombined into the recipient chromosome (for reviews, see references 15 and 44).Although reasonably well understood at this "macroscopic" level, the molecular details of the process by which DNA is transferred from donor to recipient are still being resolved. Key players are the proteins that initiate the physical transfer of ssDNA, the conjugative initiator proteins (7,22,30). These proteins introduce a site-and strand-specific ...

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Section: Resultsmentioning

confidence: 99%

The F-Plasmid TraI Protein Contains Three Functional Domains Required for Conjugative DNA Strand Transfer

Matson

Ragonese²

2005

J Bacteriol

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“…The method is based on selective precipitation of DNAprotein complexes by KCl in the presence of SDS. It has been used to show tight binding between relaxases and oriTs (17,29). Purified Mob(His 6 ) protein was incubated with supercoiled p2oriT DNA as described above, and the reactions were terminated by addition of EDTA and SDS.…”

Section: Vol 183 2001mentioning

confidence: 99%

Mobilization Function of the pBHR1 Plasmid, a Derivative of the Broad-Host-Range Plasmid pBBR1

2001

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The pBHR1 plasmid is a derivative of the small (2.6-kb), mobilizable broad-host-range plasmid pBBR1, which was isolated from the gram-negative bacterium Bordetella bronchiseptica (R. Antoine and C. Locht, Mol. Microbiol. 6:1785-1799, 1992). Plasmid pBBR1 consists of two functional cassettes and presents sequence similarities with the transfer origins of several plasmids and mobilizable transposons from gram-positive bacteria. We show that the Mob protein specifically recognizes a 52-bp sequence which contains, in addition to the transfer origin, the promoter of the mob gene. We demonstrate that this gene is autoregulated. The binding of the Mob protein to the 52-bp sequence could thus allow the formation of a protein-DNA complex with a double function: relaxosome formation and mob gene regulation. We show that the Mob protein is a relaxase, and we located the nic site position in vitro. After sequence alignment, the position of the nic site of pBBR1 corresponds with those of the nick sites of the Bacteroides mobilizable transposon Tn4555 and the streptococcal plasmid pMV158. The oriT of the latter is characteristic of a family of mobilizable plasmids that are found in gram-positive bacteria and that replicate by the rolling-circle mechanism. Plasmid pBBR1 thus appears to be a new member of this group, even though it resides in gram-negative bacteria and does not replicate via a rolling-circle mechanism. In addition, we identified two amino acids of the Mob protein necessary for its activity, and we discuss their involvement in the mobilization mechanism.

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“…plasmids (1,5,7,(13)(14)(15)(16)(17)(18)(19)(20). After the nicking reaction, VirD2 remains associated with the 5' end of the conjugative DNA strand (17)(18)(19).…”

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confidence: 99%

Agrobacterium T-strand production in vitro: sequence-specific cleavage and 5' protection of single-stranded DNA templates by purified VirD2 protein.

Jasper

Koncz

Schell

et al. 1994

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

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Virulence proteins VirDl and VirD2 are subunits of a relaxosome-like protein complex that mediates conjugational transfer of a Ti plasmid segment, the T-DNA, from Agrobacterium into higher plants. The VirDl-VirD2 complex binds to 25-bp repeats at the borders of the T-DNA and catalyzes sequence-specific nicking of the conijugative DNA strand (the T-strand) at the third base of these repeats. Nuclear localization signals present in VirD2 target the T-strand to plant ceil nuclei. In addition, VirD2 probably plays a role in the high-frequency integration of the T-DNA into the plant genome by illegitimate recombination. Whereas Agrobacterium transformation of dicots is very efficient, T-DNA integration in most monocots can barely be detected. To develop an artificial T-DNA delivery system for monocots, a technique for efficient in vitro production of T-strand DNAs was established by using VirDl and VirD2 proteins purified from overexpressing Escherichia coli strains. The topoisomerase-like VirD2 enzyme was shown to mediate precise, sequence-specific cleavage of T-DNA border sequences carried by single-stranded DNA templates, even in the absence of VirDl protein. During this reaction, VirD2 remains covalently bound to the 5' end of artificial T-strand DNAs. In contrast, VirD2, alone or in complex with VirDl, fails to nick linear double-stranded DNA templates in vitro.

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Characterization of the reaction product of the oriT nicking reaction catalyzed by Escherichia coli DNA helicase I

Cited by 53 publications

References 30 publications

The F-Plasmid TraI Protein Contains Three Functional Domains Required for Conjugative DNA Strand Transfer

The F-Plasmid TraI Protein Contains Three Functional Domains Required for Conjugative DNA Strand Transfer

Mobilization Function of the pBHR1 Plasmid, a Derivative of the Broad-Host-Range Plasmid pBBR1

Agrobacterium T-strand production in vitro: sequence-specific cleavage and 5' protection of single-stranded DNA templates by purified VirD2 protein.

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