Concomitant Reconstitution of TraI-catalyzed DNA Transesterase and DNA Helicase Activity in Vitro

Csitkovits, Vanessa C.; Đermić, Damir; Zechner, Ellen L.

doi:10.1074/jbc.m407970200

Cited by 12 publications

(30 citation statements)

References 42 publications

(31 reference statements)

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“…Although binding of the relaxase at oriT requires limited denaturation of the DNA around nic, the extent of the melted region does not appear to be enough to support helicase activity (16,46). Unwinding the DNA is an activity of the helicase domain of TraI in the F-plasmid (29), which requires over 30 nucleotides of single-stranded DNA upstream of the 5Ј end of the nic site in vivo (7). Therefore, a "mating signal" must be produced by donor-recipient cell contact, resulting in localized melting at oriT that initiates unwinding by TraI.…”

Section: Discussionmentioning

confidence: 99%

Mutations in the C-Terminal Region of TraM Provide Evidence for In Vivo TraM-TraD Interactions during F-Plasmid Conjugation

Frost

2005

J Bacteriol

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Conjugation is a major mechanism for disseminating genetic information in bacterial populations, but the signal that triggers it is poorly understood in gram-negative bacteria. F-plasmid-mediated conjugation requires TraM, a homotetramer, which binds cooperatively to three binding sites within the origin of transfer. Using in vitro assays, TraM has previously been shown to interact with the coupling protein TraD. Here we present evidence that F conjugation also requires TraM-TraD interactions in vivo. A three-plasmid system was used to select mutations in TraM that are defective for F conjugation but competent for tetramerization and cooperative DNA binding to the traM promoter region. One mutation, K99E, was particularly defective in conjugation and was further characterized by affinity chromatography and coimmunoprecipitation assays that suggested it was defective in interacting with TraD. A C-terminal deletion (S79*, where the asterisk represents a stop codon) and a missense mutation (F121S), which affects tetramerization, also reduced the affinity of TraM for TraD. We propose that the C-terminal region of TraM interacts with TraD, whereas its N-terminal domain is involved in DNA binding. This arrangement of functional domains could in part allow TraM to receive the mating signal generated by donor-recipient contact and transfer it to the relaxosome, thereby triggering DNA transfer.

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Section: Discussionmentioning

confidence: 99%

Mutations in the C-Terminal Region of TraM Provide Evidence for In Vivo TraM-TraD Interactions during F-Plasmid Conjugation

Frost

2005

J Bacteriol

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“…In the bacterial cell, recruitment and activation of the conjugative helicase occur not on naked DNA but within an initiator complex called the relaxosome (67). For the F-like plasmid R1, sequence-specific DNA binding properties of the plasmid proteins TraI, TraY, TraM, and the host integration factor (IHF) direct assembly of the relaxosome at oriT (10,12,29,33,51,52). Integration of protein TraM confers recognition features to the relaxosome, which permit its selective docking to TraD, the coupling protein associated with the conjugative type IV secretion system (T4CP) (2,15,49).…”

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confidence: 99%

“…The extraordinary efficiency of these proteins in intercellular DNA strand transfer belies this prediction and instead hints strongly at a coordinated progression of the initiation pathway. Since relaxosome assembly is thus far insufficient to initiate helicase activity on supercoiled oriT substrates in vitro, we have developed a series of heteroduplex DNA substrates which support the unwinding reaction and model possible intermediate structures of R1 plasmid strand transfer initiation (10). In this system linear double-stranded DNA (dsDNA) substrates with a central region of sequence heterogeneity trap defined lengths of R1 oriT sequence in unwound conformation.…”

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confidence: 99%

Protein and DNA Effectors Control the TraI Conjugative Helicase of Plasmid R1

et al. 2009

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Controlled duplex DNA unwinding is a crucial prerequisite for the expression and maintenance of genomes. Genomemanipulating and -regulating proteins are central to that biological function in recognizing appropriate DNA targets at initiation sequences and unwinding the complementary strands to provide single-stranded DNA (ssDNA) templates for nucleic acid synthesis and other processing reactions. The protein machineries involved include nucleic acid helicases. DNA helicases are powerful enzymes that convert the energy of nucleoside triphosphate hydrolysis to directional DNA strand translocation and separation of the double helix into its constituent single strands (for reviews, see references 13, 14, 16, 38, 55, and 64). By necessity, these enzymes interact with DNA strands via mechanisms independent of sequence recognition. At replication initiation helicases gain controlled access to the doublestranded genome at positions determined by the DNA binding properties of initiator proteins that comprise an origin recognition complex (1,9,17,31,45,66). The mechanisms supporting localized unwinding within the complex include initiatorinduced DNA looping, wrapping, and bending and feature regions of low thermodynamic stability. The exposed ssDNA mediates helicase binding followed by directional translocation along that strand until the enzyme engages the duplex for unwinding.In the MOB F family of conjugation systems, the plasmid DNA strand destined for transfer (T strand) is unwound from its complement by a dedicated conjugative helicase, TraI of F-like plasmids or TrwC of the IncW paradigm. These enzymes are remarkable in that the same polypeptides additionally harbor in a distinct domain a DNA transesterase activity. That function is required to recognize and cleave the precise phosphodiester bond, nic, in the T strand where unwinding of the secretion substrate begins. In current models the conjugative helicases are thus targeted to the transfer origin (oriT) of their cognate plasmid by the high-affinity DNA sequence interactions of their N-terminal DNA transesterase domains. In the bacterial cell, recruitment and activation of the conjugative helicase occur not on naked DNA but within an initiator complex called the relaxosome (67). For the F-like plasmid R1, sequence-specific DNA binding properties of the plasmid proteins TraI, TraY, TraM, and the host integration factor (IHF) direct assembly of the relaxosome at oriT (10,12,29,33,51,52). Integration of protein TraM confers recognition features to the relaxosome, which permit its selective docking to TraD, the coupling protein associated with the conjugative type IV secretion system (T4CP) (2,15,49). In current models, the T4CP forms a hexameric translocation pore at the cytoplasmic membrane that not only governs substrate entry to the envelope spanning type IV secretion machinery but also provides energy for macromolecular transport via ATP hydrolysis (36,50). These models propose that T4CPs provide not only a physical bridge between the plasmid and the type IV ...

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“…The hypothetical protein encoded by ORF75419 (760 aa) contained two domains with putative DNA helicase function, between amino acid positions 112 and 186 (DExH box, 53.8% alignment without gaps) and 599 and 721 (HELICc motif, 90.8% alignment without gaps). Helicases are implicated in the unwinding of the nicked plasmid in relaxosome formation during conjugation (13,37). Thirteen highly identical orthologs (49% to 99%) were currently detected in the NCBI database for the ORF75419 peptide by BLAST searches.…”

Section: Resultsmentioning

confidence: 99%

TheclcElement ofPseudomonassp. Strain B13, a Genomic Island with Various Catabolic Properties

et al. 2006

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Pseudomonas sp. strain B13 is a bacterium known to degrade chloroaromatic compounds. The properties to use 3-and 4-chlorocatechol are determined by a self-transferable DNA element, the clc element, which normally resides at two locations in the cell's chromosome. Here we report the complete nucleotide sequence of the clc element, demonstrating the unique catabolic properties while showing its relatedness to genomic islands and integrative and conjugative elements rather than to other known catabolic plasmids. As far as catabolic functions, the clc element harbored, in addition to the genes for chlorocatechol degradation, a complete functional operon for 2-aminophenol degradation and genes for a putative aromatic compound transport protein and for a multicomponent aromatic ring dioxygenase similar to anthranilate hydroxylase. The genes for catabolic functions were inducible under various conditions, suggesting a network of catabolic pathway induction. For about half of the open reading frames (ORFs) on the clc element, no clear functional prediction could be given, although some indications were found for functions that were similar to plasmid conjugation. The region in which these ORFs were situated displayed a high overall conservation of nucleotide sequence and gene order to genomic regions in other recently completed bacterial genomes or to other genomic islands. Most notably, except for two discrete regions, the clc element was almost 100% identical over the whole length to a chromosomal region in Burkholderia xenovorans LB400. This indicates the dynamic evolution of this type of element and the continued transition between elements with a more pathogenic character and those with catabolic properties.

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Concomitant Reconstitution of TraI-catalyzed DNA Transesterase and DNA Helicase Activity in Vitro

Cited by 12 publications

References 42 publications

Mutations in the C-Terminal Region of TraM Provide Evidence for In Vivo TraM-TraD Interactions during F-Plasmid Conjugation

Mutations in the C-Terminal Region of TraM Provide Evidence for In Vivo TraM-TraD Interactions during F-Plasmid Conjugation

Protein and DNA Effectors Control the TraI Conjugative Helicase of Plasmid R1

TheclcElement ofPseudomonassp. Strain B13, a Genomic Island with Various Catabolic Properties

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