Extent of Single-stranded DNA Required for Efficient TraI Helicase Activity in Vitro

Csitkovits, Vanessa C.; Zechner, Ellen L.

doi:10.1074/jbc.m310025200

Cited by 15 publications

(19 citation statements)

References 53 publications

(57 reference statements)

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“…Potassium permanganate mapping of the protein-induced topological change on supercoiled oriT DNA in vitro revealed a highly specific opening of 2 to 3 bp at nic that was mediated by TraI alone or in the reconstituted relaxosome. In contrast single effectors and their combinations did not result in localized melting anywhere else in the entire oriT region that would be compatible with the ssDNA length requirements previously defined for detectable TraI helicase activity (16,17). The simplest explanation for the lack of progression of the reconstituted preinitiation complex to a complex competent for T-strand unwinding would be that the torsional strain induced during assembly of this initial complex in vitro is distributed generally over the plasmid and does not force extended duplex melting at nic-adjacent sequences.…”

Section: Discussionmentioning

confidence: 90%

Plasmid R1 Conjugative DNA Processing Is Regulated at the Coupling Protein Interface

et al. 2009

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Selective substrate uptake controls initiation of macromolecular secretion by type IV secretion systems in gram-negative bacteria. Type IV coupling proteins (T4CPs) are essential, but the molecular mechanisms governing substrate entry to the translocation pathway remain obscure. We report a biochemical approach to reconstitute a regulatory interface between the plasmid R1 T4CP and the nucleoprotein relaxosome dedicated to the initiation stage of plasmid DNA processing and substrate presentation. The predicted cytosolic domain of T4CP TraD was purified in a predominantly monomeric form, and potential regulatory effects of this protein on catalytic activities exhibited by the relaxosome during transfer initiation were analyzed in vitro. Type IV secretion systems (T4SS) in gram-negative bacteria mediate translocation of macromolecules out of the bacterial cell (14). The transmission of effector proteins and DNA into plant cells or other bacteria via cell-cell contact is one example of their function, and conjugation systems as well as the transferred DNA (T-DNA) delivery system of the phytopathogen Agrobacterium tumefaciens are prototypical of the T4SS family. Macromolecular translocation is achieved by a membranespanning protein machinery comprised of 12 gene products, VirB1 to VirB11 and an associated factor known as the coupling protein (VirD4) (66). The T4SS-associated coupling protein (T4CP) performs a crucial function in recognition of appropriate secretion substrates and governing entry of those molecules to the translocation pathway (7,8,10,30,41). In conjugation systems substrate recognition is applied to the relaxosome, a nucleoprotein complex of DNA transfer initiator proteins assembled specifically at the plasmid origin of transfer (oriT). In current models, initiation of the reactions that provide the single strand of plasmid (T-strand) DNA for secretion to recipient bacteria is expected to resemble the initiation of chromosomal replication (for reviews, see references 18, 54, and 81). Controlled opening of the DNA duplex is required to permit entry of the DNA processing machinery. The task of remodeling the conjugative oriT is generally ascribed to two or three relaxosome auxiliary factors, of host and plasmid origin, which occupy specific DNA binding sites at this locus. Intrinsic to the relaxosome is also a site-and strand-specific DNA transesterase activity that breaks the phosphodiester backbone at nic (5). Upon cleavage, the transesterase enzyme (also called relaxase) forms a reversible phosphotyrosyl linkage to the 5Ј end of the DNA. Duplex unwinding initiating from this site produces the single-stranded T strand to be exported. A wealth of information is available supporting the importance of DNA sequence recognition and binding by relaxosome components at oriT to the transesterase reaction in vitro and for effective conjugative transfer (for reviews, see references 18, 54, and 81). On the other hand, the mechanisms controlling release of the 3Ј-OH generated at nic and the subsequent DNA unwind...

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Section: Discussionmentioning

confidence: 90%

Plasmid R1 Conjugative DNA Processing Is Regulated at the Coupling Protein Interface

et al. 2009

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“…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. Unexpectedly, efficient helicase activity initiated from a melted oriT duplex required ssDNA twice as long (60 nt) as that previously observed on substrates lacking this sequence (11).…”

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confidence: 71%

“…The enzyme requires ssDNA 5Ј to the duplex junction (32), and a minimum length of 30 nucleotides (nt) is necessary to promote efficient duplex unwinding on substrates lacking oriT (11,54). To our knowledge, oriT is the only sequence where the helicase activity is naturally initiated, however.…”

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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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“…Typical superfamily I helicases, such as UvrD and RecD, can efficiently unwind a duplex DNA with a singlestranded tail of 10 -12 nucleotides (34,35). However, TraI requires at least a 27-nucleotide tail for efficient unwinding and cannot unwind duplex DNA if the tail is shorter than 20 nucleotides (36). Quantitative analysis in this work suggests that the unusually long single-stranded tail required by TraI for DNA unwinding arises from its unique ssDNA-binding properties.…”

Section: Discussionmentioning

confidence: 85%

Functional Characterization of the Multidomain F Plasmid TraI Relaxase-Helicase

Cheng¹,

McNamara²,

Miley³

et al. 2011

Journal of Biological Chemistry

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TraI, a bifunctional enzyme containing relaxase and helicase activities, initiates and drives the conjugative transfer of the Escherichia coli F plasmid. Here, we examined the structure and function of the TraI helicase. We show that TraI binds to singlestranded DNA (ssDNA) with a site size of ϳ25 nucleotides, which is significantly longer than the site size of other known superfamily I helicases. Low cooperativity was observed with the binding of TraI to ssDNA, and a double-stranded DNA-binding site was identified within the N-terminal region of TraI 1-858, outside the core helicase motifs of TraI. We have revealed that the affinity of TraI for DNA is negatively correlated with the ionic strength of the solution. The binding of AMPPNP or ADP results in a 3-fold increase in the affinity of TraI for ssDNA. Moreover, TraI prefers to bind ssDNA oligomers containing a single type of base. Finally, we elucidated the solution structure of TraI using small angle x-ray scattering. TraI exhibits an ellipsoidal shape in solution with four domains aligning along one axis. Taken together, these data result in the assembly of a model for the multidomain helicase activity of TraI.Conjugative plasmid transfer is a central mechanism for the horizontal exchange of genetic material between bacterial cells, as well as for the spread of antibiotic resistance genes and virulence factors (1-3). Conjugative plasmid transfer requires both a DNA relaxase and a DNA helicase. The relaxase initiates DNA transfer by cleaving the transferred strand at a specific site within the oriT, forming a 5Ј-phosphotyrosine intermediate. Following nicking, the helicase uses the energy from ATP hydrolysis to unwind the plasmid and drive the transfer of DNA into the recipient cell. The relaxase completes plasmid transfer by breaking the covalent phosphotyrosine linkage and releasing the transferred DNA for replication in the recipient (2, 4, 5).

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Extent of Single-stranded DNA Required for Efficient TraI Helicase Activity in Vitro

Cited by 15 publications

References 53 publications

Plasmid R1 Conjugative DNA Processing Is Regulated at the Coupling Protein Interface

Plasmid R1 Conjugative DNA Processing Is Regulated at the Coupling Protein Interface

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

Functional Characterization of the Multidomain F Plasmid TraI Relaxase-Helicase

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