Deletion analysis of the F plasmid oriT locus

Fu, Yang; Tsai, Mei-Mei; Luo, Yanan; Deonier, Richard C.

doi:10.1128/jb.173.3.1012-1020.1991

Cited by 42 publications

(50 citation statements)

References 25 publications

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“…Vertical arrows indicate the end-point of progressive deletions from the right with numbers showing the folddecrease in transfer efficiency. A deletion extending to the double asterisk is transfer and nick null (Fu et al, 1991;Luo et al, 1994). Horizontal arrows indicate inverted or direct repeats.…”

Section: The Rp4 Relaxosomementioning

confidence: 99%

Nicking by transesterification: the reaction catalysed by a relaxase

Byrd¹,

Matson

1997

Molecular Microbiology

108

117

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SummaryDNA relaxases play an essential role in the initiation and termination of conjugative DNA transfer. Purification and characterization of relaxases from several plasmids has revealed the reaction mechanism: relaxases nick duplex DNA in a site-and strand-specific manner by catalysing a transesterification. The product of the reaction is a nicked double-stranded DNA molecule with a sequestered 3Ј-OH and the relaxase covalently bound to the 5Ј end of the cleaved strand via a phosphotyrosyl linkage. The relaxase-catalysed transesterification is isoenergetic and reversible; a second transesterification ligates the nicked DNA. However, the covalent nucleoprotein complex is relatively long-lived, a property that is likely to be essential for its role as an intermediate in the process of conjugative DNA transfer. Subsequent unwinding of the nicked DNA intermediate is required to produce the single strand of DNA transferred to the recipient cell. This reaction is catalysed by a DNA helicase, an activity intrinsic to the relaxase protein in some, but not all, plasmid systems. The first relaxase-catalysed transesterification is essential for initiation of conjugative strand transfer, whereas the second is presumably required for termination of the process. The relaxase, in conjunction with several auxiliary proteins, forms the relaxation complex or relaxosome first described nearly 30 years ago as being associated with conjugative and mobilizable plasmids.

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Section: The Rp4 Relaxosomementioning

confidence: 99%

Nicking by transesterification: the reaction catalysed by a relaxase

Byrd¹,

Matson

1997

Molecular Microbiology

108

117

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“…The third binding site with the lowest affinity for TraM (sbmC) as determined by DNase I footprinting is located between TraY (sbyA) and the IHF binding site (IHFB) close to oriT. Deletion of smbC shows the strongest effect on DNA transfer (9). Di Laurenzio and coworkers (6) suggest that the binding to sbmC may be crucial in the DNA transfer process because TraM could signal the cell that mating pair formation is completed and DNA transfer can be initiated.…”

mentioning

confidence: 99%

“…The TraD protein with a molecular mass of about 82 kDa is located in the inner membrane (12,20) and was shown to bind to DNA cellulose, indicating nonspecific binding to nucleic acids (20). The amino acid sequence deduced from the nucleotide sequence contains an ATP-binding motif (9) and preliminary data suggest a DNA-dependent ATPase activity of TraD (20).…”

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confidence: 99%

The cytoplasmic DNA-binding protein TraM binds to the inner membrane protein TraD in vitro

Disqué-Kochem

Dreiseikelmann

1997

J Bacteriol

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The cytoplasmic protein TraM is one of four essential gene products of the F factor which are involved in DNA transfer after mating pair formation. TraM binds to three specific sites within the oriT region. Besides regulation of its own synthesis, the precise function of TraM during conjugation is not yet known. In the present work, the affinity of TraM to TraD was studied in vitro by an overlay assay and by affinity chromatography. Whether the interaction between TraM and TraD causes a transient or permanent anchoring of the F factor to the site of transfer is discussed. A 35-kDa host membrane protein of yet unknown function also shows affinity to TraM and may be involved in this anchoring process as well.Bacterial conjugation is a process during which DNA is transferred from a donor to a recipient across the envelope of both cells. One of the best-studied conjugative plasmids is the F factor of Escherichia coli (for review, see references 7, 8, 11, and 27). After pilus synthesis and mating pair formation during the conjugation of the F factor, four additional gene products of the tra operon are directly involved in a successful DNA translocation across the envelope of the donor. These products are encoded by the genes traI, traY, traD, and traM. Of these four essential Tra proteins, TraI is the best characterized. The TraI protein, also called helicase I, catalyzes the strand-and site-specific nicking of the F factor at oriT (16,22). Furthermore, TraI unwinds duplex DNA in an ATP-hydrolysis-dependent reaction (1-3). The precise functions of TraY, TraD, and TraM are not yet known. It was recently shown in vivo that the nicking reaction of TraI is stimulated by TraY and the integration host factor (IHF) of E. coli (18). Both proteins bend DNA when bound to specific sites within the oriT region (14,15,17,19,26). Nelson and coworkers (18) propose that TraY and IHF form a nucleoprotein complex with oriT DNA which consequently can be recognized and nicked by TraI more efficiently.While TraI and TraM function in the processing of the F DNA, TraD seems to be a component of the DNA transfer apparatus. It is generally accepted that export of the singlestranded F DNA across the envelope of the donor cell proceeds through a complex pore formed by or with participation of various Tra proteins. One of these proteins is probably TraD. The TraD protein with a molecular mass of about 82 kDa is located in the inner membrane (12,20) and was shown to bind to DNA cellulose, indicating nonspecific binding to nucleic acids (20). The amino acid sequence deduced from the nucleotide sequence contains an ATP-binding motif (9) and preliminary data suggest a DNA-dependent ATPase activity of TraD (20).TraM is a cytoplasmic protein with a molecular mass of 14.5 kDa. In the past, investigations of the TraM protein have mainly been focused on its regulatory function. Maximal traM transcription requires TraY in addition to expression of the tra operon. TraM binds to three sites within the oriT region of F (6). Two sites with high affinity fo...

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“…The F oriT sequence is located within an approximately 250-bp segment at one end of the transfer region (5,10). Within the oriT sequence, the binding sites for F-encoded TraY and TraM and for integration host factor (IHF) are situated near the nick site that is recognized by F TraI protein.…”

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confidence: 99%

Initiation and Termination of DNA Transfer during Conjugation of IncI1 Plasmid R64: Roles of Two Sets of Inverted Repeat Sequences within oriT in Termination of R64 Transfer

Furuya

Komano

2000

J Bacteriol

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Intercellular transfer of plasmid DNA during bacterial conjugation initiates and terminates at a specific origin of transfer, oriT. We have investigated the oriT structure of conjugative plasmid R64 with regard to the initiation and termination of DNA transfer. Using recombinant plasmids containing two tandemly repeated R64 oriT sequences with or without mutations, the subregions required for initiation and termination were determined by examining conjugation-mediated deletion between the repeated oriTs. The oriT subregion required for initiation was found to be identical to the 44-bp oriT core sequence consisting of two units, the conserved nick region sequence and the 17-bp repeat A sequence, that are recognized by R64 relaxosome proteins NikB and NikA, respectively. In contrast, the nick region sequence and two sets of inverted repeat sequences within the 92-bp minimal oriT sequence were required for efficient termination. Mutant repeat A sequences lacking NikA-binding ability were found to be sufficient for termination, suggesting that the inverted repeat structures are involved in the termination process. A duplication of the DNA segment between the repeated oriTs was also found after mobilization of the plasmid carrying initiation-deficient but terminationproficient oriT and initiation-proficient but termination-deficient oriT, suggesting that the 3 terminus of the transferred strand is elongated by rolling-circle-DNA synthesis.Intercellular transfer of plasmid DNA during bacterial conjugation is accomplished by the function of transfer genes borne on each conjugative plasmid (for reviews, see references 4 and 15). All conjugative and mobilizable plasmids, such as R64, F, RP4, and R1162, contain oriT sites as cis elements which function as the origin of transfer of plasmid DNA. At the initiation stage of DNA transfer, a site-and strand-specific nick is introduced into the oriT site with a covalent attachment of the cognate relaxase protein to the 5Ј terminus of the nicked strand. The nicked strand is transferred from donor to recipient cells with the 5Ј terminus leading through a putative channel. In the donor cells, replacement strand DNA synthesis reconstitutes the double-stranded plasmid DNA. After one round of DNA transfer, the relaxase-attached 5Ј terminus of the transferred-strand DNA is religated to its 3Ј terminus to reconstitute the circular structure, and complementary-strand synthesis establishes double-stranded plasmid DNA in the recipient cells. Among these steps, the mechanisms by which initiation and termination of conjugative DNA transfer occur are important issues which remain to be elucidated.Initiation and termination of conjugative DNA transfer at the oriT site were extensively studied using a small mobilizable plasmid, R1162 (RSF1010). R1162 oriT consists of a specific nick site and a 10-bp inverted repeat with one mismatch, which is situated 8 bp from the nick site (3, 27). Three R1162 proteins, MobA, MobB, and MobC, form a protein-DNA complex called the relaxosome at oriT (27). From ...

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Deletion analysis of the F plasmid oriT locus

Cited by 42 publications

References 25 publications

Nicking by transesterification: the reaction catalysed by a relaxase

Nicking by transesterification: the reaction catalysed by a relaxase

The cytoplasmic DNA-binding protein TraM binds to the inner membrane protein TraD in vitro

Initiation and Termination of DNA Transfer during Conjugation of IncI1 Plasmid R64: Roles of Two Sets of Inverted Repeat Sequences within oriT in Termination of R64 Transfer

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