Recognition and processing of the origin of transfer DNA by conjugative relaxase TrwC

Abstract

Relaxases are DNA strand transferases that catalyze the initial and final stages of DNA processing during conjugative cell-to-cell DNA transfer. Upon binding to the origin of transfer (oriT) DNA, relaxase TrwC melts the double helix. The three-dimensional structure of the relaxase domain of TrwC in complex with its cognate DNA at oriT shows a fold built on a two-layer alpha/beta sandwich, with a deep narrow cleft that houses the active site. The DNA includes one arm of an extruded cruciform, an essential feature for specific recognition. This arm is firmly embraced by the protein through a beta-ribbon positioned in the DNA major groove and a loop occupying the minor groove. It is followed by a single-stranded DNA segment that enters the active site, after a sharp U-turn forming a hydrophobic cage that traps the N-terminal methionine. Structural analysis combined with site-directed mutagenesis defines the architecture of the active site.

The mating pair formation system of conjugative plasmids—A versatile secretion machinery for transfer of proteins and DNA

“…The crystal structures of the relaxase domains of TrwC (R388) and TraI (F) have been determined (Datta et al, 2003;Guasch et al, 2003). They reveal a conserved compact molecular scaVold possessing features that explain the high aYnity and speciWcity for their respective DNA substrates.…”

“…The structure consists of a Wve-strand antiparallel -sheet core ('palm' domain), which is Xanked by several -helices. The structure of TrwC in complex with a 25-mer oligonucleotide corresponding to the sequence of the transfer strand just upstream of the nic site furthermore provides details of the interactions between the relaxase and its ssDNA substrate (Guasch et al, 2003). This structure corresponds to the situation most probably encountered after cleavage at the nic site.…”

“…Possible roles for the metal cation consist in polarizing the scissile phosphate of the ssDNA substrate for facilitating the nucleophilic attack by the catalytic tyrosine hydroxyl group, and of stabilizing the ensuing transition state. An aspartate residue in TrwC has been proposed as a candidate general base that could activate the catalytic tyrosine by proton abstraction (Guasch et al, 2003). TraI and TrwC each contain a second tyrosine residue which is thought to catalyze the second DNA strand transfer reaction that recircularizes the plasmid after one round of transfer (Grandoso et al, 2000;Pansegrau and Lanka, 1996b).…”

Agrobacterium tumefaciens VirB6 Domains Direct the Ordered Export of a DNA Substrate Through a Type IV Secretion System

“…[5][6][7][8] Finally, the T4CP functions together with a trans-envelope secretion channel composed of the mating pair formation (Mpf) proteins to deliver the DNA transfer intermediate to the cell exterior. 9 The processing and recruitment reactions are biochemically well characterized for several conjugation systems, and recent structurefunction studies have been aided by available crystal structures for the TrwC 10,11 and TraI 12,13 relaxases of plasmids R388 and F, respectively, and TrwB T4CP 14 of plasmid R388. Until recently, however, there has been very little understanding of the secretion channel architecture or mechanism of action.…”

Hepatitis C virus biology

“…A similar mechanism has been proposed for the integration of a bacterial virus, bacteriophage CTXȠ, into the Vibrio cholerae genome 9 . Moreover, related models would also explain other systems involved in horizontal gene transfer, including plasmid and viral replication (REP proteins), conjugal plasmid transfer from cell to cell (relaxases) and certain transposase enzymes [10][11][12][13][14] .…”