Strand Selection During Bacterial Mating

Wd, Rupp; Ihler, Garret M.

doi:10.1101/sqb.1968.033.01.073

Cited by 79 publications

(32 citation statements)

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“…It has no free end and is therefore protected from exonuclease attack and TraI is suitably oriented to catalyse a second transesterification that results in the termination of strand transfer. This would also be consistent with an overall 5Ј to 3Ј polarity of transfer of genetic markers from an Hfr strain (Ohki and Tomizawa, 1968;Rupp and Ihler, 1968), as DNA sequences located at the 5Ј end of the transferred strand would enter the recipient cell first. Confirmation of this view awaits complete reconstitution of nicking/unwinding in vitro, which has not yet been achieved for any system.…”

Section: Relaxosome Assemblysupporting

confidence: 67%

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: Relaxosome Assemblysupporting

confidence: 67%

Nicking by transesterification: the reaction catalysed by a relaxase

Byrd¹,

Matson

1997

Molecular Microbiology

108

117

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“…It has long been known that the nicked DNA strand enters the recipient cell with a 5'-to-3' polarity during the process of bacterial conjugation (16,20). Presumably, the end is blocked in some way to protect the transferred DNA from attack by exonucleases within the recipient cell.…”

Section: Methodsmentioning

confidence: 99%

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

1993

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DNA helicase I, encoded on the Escherichia coli F plasmid, catalyzes a site-and strand-specific nicking reaction within the F plasmid origin of transfer (oril) to initiate conjugative DNA strand transfer. The product of the nicking reaction contains a single phosphodiester bond interruption as determined by single-nucleotide resolution mapping of both sides of the nick site. This analysis has demonstrated that the nick is located at precisely the same site previously shown to be nicked in vivo (T. L. Thompson, M. B. Centola, and R. C.Deonier, J. Mol. Biol. 207:505-512, 1989). In addition, studies with two oriT point mutants have confirmed the specificity of the in vitro reaction. Characterization of the nicked DNA product has revealed a modified 5' end and a 3' OH available for extension by E. coli DNA polymerase I. Precipitation of nicked DNA with cold KC1 in the presence of sodium dodecyl sulfate suggests the existence of protein covalently attached to the nicked DNA molecule. The covalent nature of this interaction has been directly demonstrated by transfer of radiolabeled phosphate from DNA to protein. On the basis of these results, we propose that helicase I becomes covalently bound to the 5' end of the nicked DNA strand as part of the reaction mechanism for phosphodiester bond cleavage. A model is presented to suggest how helicase I could nick the F plasmid at oriT and subsequently unwind the duplex DNA to provide single-stranded DNA for strand transfer during bacterial conjugation.

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“…2 TraI with the DNA helicase activity in the direction from 5 0 to 3 0 unwinds double-stranded DNA from the nick introduced. The single-stranded DNA so generated is thought to be transferred to the recipient cell in the direction from 5 0 to 3 0 (Ohki & Tomizawa 1968;Cohen et al 1968;Rupp & Ihler 1968). Note that the 5 0 end of the single-stranded DNA to be transferred is attached with TraI, and that any proteins including TraI are not transferred by conjugation (Silver et al 1965;Rees & Wilkins 1990).…”

Section: The Rejoining Reaction Involved In Termination Of Conjugal Dmentioning

confidence: 99%

“…The site-and strand-specific nicking is thought to be the initiation reaction of DNA transfer promoted by a conjugative plasmid; nicking facilitates the production of the single-stranded DNA, which is transferred to the recipient cell in the 5 0 to 3 0 direction (Ohki & Tomizawa 1968;Cohen et al 1968;Rupp & Ihler 1968) by the action of TraI with the DNA helicase activity unwinding the double-stranded DNA in the direction from 5 0 to 3 0 (Lahue & Matson 1988;Fukuda & Ohtsubo 1995). 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).…”

Section: Introductionmentioning

confidence: 99%