Structural Insights into Single-Stranded DNA Binding and Cleavage by F Factor TraI

Datta, Saumen; Larkin, Chris; Schildbach, Joel F.

doi:10.1016/j.str.2003.10.001

Cited by 90 publications

(120 citation statements)

References 56 publications

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“…Diffraction data were indexed and scaled with HKL2000 and MOSFLM (CCP4) (65). Initial phases were determined by molecular replacement in Molrep (CCP4) (65) with apo-TraI [Protein Data Bank ID code 1P4D (41)] as a search model (SI Table 1). The Protein Data Bank ID codes for the structures without and with PNP are 2Q7T and 2Q7U, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…The relaxase completes DNA transfer by reversing the covalent phosphotyrosine linkage and releasing the T-strand. In the F plasmid, this relaxase is located in the N-terminal domain of a large multifunctional protein, TraI (DNA helicase I) (22,23,(26)(27)(28) (38)(39)(40)(41)]. Optimal relaxase cleavage, ligation, and transfer of ssDNA require the metal ion and two catalytic tyrosines, one from each pair (42).…”

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confidence: 99%

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Disrupting antibiotic resistance propagation by inhibiting the conjugative DNA relaxase

Lujan

Guogas²,

Ragonese³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Conjugative transfer of plasmid DNA via close cell-cell junctions is the main route by which antibiotic resistance genes spread between bacterial strains. Relaxases are essential for conjugative transfer and act by cleaving DNA strands and forming covalent phosphotyrosine linkages. Based on data indicating that multityrosine relaxase enzymes can accommodate two phosphotyrosine intermediates within their divalent metal-containing active sites, we hypothesized that bisphosphonates would inhibit relaxase activity and conjugative DNA transfer. We identified bisphosphonates that are nanomolar inhibitors of the F plasmid conjugative relaxase in vitro. Furthermore, we used cell-based assays to demonstrate that these compounds are highly effective at preventing DNA transfer and at selectively killing cells harboring conjugative plasmids. Two potent inhibitors, clodronate and etidronate, are already clinically approved to treat bone loss. Thus, the inhibition of conjugative relaxases is a potentially novel antimicrobial approach, one that selectively targets bacteria capable of transferring antibiotic resistance and generating multidrug resistant strains.antimicrobial ͉ bacterial conjugation ͉ bisphosphonates ͉ F plasmid TraI ͉ relaxase inhibition C onjugative elements are responsible for the majority of horizontal gene transfers within and between bacterial strains (reviewed in ref. 1), as first described for the Escherichia coli F plasmid by Lederberg and Tatum in 1946 (2). Conjugative DNA transfer is also the central mechanism by which antibiotic resistance and virulence factors are propagated in bacterial populations (reviewed in ref.3). Indeed, it is well established that antibiotic resistance can be rapidly acquired in clinical settings and that such acquisition is critically dependent on conjugative DNA transfer (reviewed in ref. 4). Small-molecule inhibition of conjugation could prove to be a powerful method for curbing the generation and spread of multidrug-resistant strains. Past studies suggested that various antibiotics, polycyclic chemicals, and crude extracts inhibit conjugation at concentrations less than the antibacterial minimum inhibitory concentration (5-11); however, most of these effects have been attributed to nonconjugation-specific inhibition of bacterial growth or DNA synthesis (12)(13)(14)(15). This study describes a bottom-up approach used to identify the first small molecule inhibitors of conjugative DNA transfer that target an enzyme of the conjugative system.The DNA relaxase is a central enzyme in each conjugative system (16-18) and thus is a prime target for inhibition. The conjugative relaxase initiates DNA transfer with a site-and strand-specific ssDNA nick in the transferred strand (T-strand) at the origin of transfer (oriT), forming a covalent 5Ј-phosphotyrosine intermediate (16,(19)(20)(21)(22)(23). The nicked T-strand moves from the donor cell (plasmid ϩ ) to the recipient cell (plasmid Ϫ ) via an intercellular junction mediated by a type IV secretion system (reviewed in refs. 19, 24,...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Disrupting antibiotic resistance propagation by inhibiting the conjugative DNA relaxase

Lujan

Guogas²,

Ragonese³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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“…Because R150 and E187 have opposite charges and Ala substitutions for them cause distinct effects on the fluorophore's behavior, one possibility is that altered electrostatic interactions between fluorophore and protein may explain the altered fluorescent characteristic. An examination of the TraI36 structure (Datta et al, 2003), however, shows that residues 150 and 187 are located nearly 30 Å apart, making a model involving direct charge-charge interactions seem less likely.…”

Section: Designing the Oligonucleotidementioning

confidence: 99%

Chapter 12 Using Fluorophore-Labeled Oligonucleotides to Measure Affinities of Protein–DNA Interactions

Anderson

Larkin

Guja

et al. 2008

Methods in Enzymology

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Changes in fluorescence emission intensity and anisotropy can reflect changes in the environment and molecular motion of a fluorophore. Researchers can capitalize on these characteristics to assess the affinity and specificity of DNA-binding proteins using fluorophore-labeled oligonucleotides. While there are many advantages to measuring binding using fluorescent oligonucleotides, there are also some distinct disadvantages. Here we describe some of the relevant issues for the novice, illustrating key points using data collected with the F plasmid relaxase domain and a variety of labeled oligonucleotides. Topics include selection of a fluorophore, experimental design using a fluorometer equipped with an automatic titrating unit, and analysis of direct binding and competition assays.

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“…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.…”

Section: Conjugative Relaxases: Dna Carrier Proteins Secreted By the mentioning

confidence: 99%

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

Schröder

Lanka

2005

Plasmid

188

146

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The mating pair formation (Mpf) system functions as a secretion machinery for intercellular DNA transfer during bacterial conjugation. The components of the Mpf system, comprising a minimal set of 10 conserved proteins, form a membrane-spanning protein complex and a surface-exposed sex pilus, which both serve to establish intimate physical contacts with a recipient bacterium. To function as a DNA secretion apparatus the Mpf complex additionally requires the coupling protein (CP). The CP interacts with the DNA substrate and couples it to the secretion pore formed by the Mpf system. Mpf/CP conjugation systems belong to the family of type IV secretion systems (T4SS), which also includes DNA-uptake and -release systems, as well as eVector protein translocation systems of bacterial pathogens such as Agrobacterium tumefaciens (VirB/VirD4) and Helicobacter pylori (Cag). The increased eVorts to unravel the molecular mechanisms of type IV secretion have largely advanced our current understanding of the Mpf/CP system of bacterial conjugation systems. It has become apparent that proteins coupled to DNA rather than DNA itself are the actively transported substrates during bacterial conjugation. We here present a uniWed and updated view of the functioning and the molecular architecture of the Mpf/CP machinery. 

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Structural Insights into Single-Stranded DNA Binding and Cleavage by F Factor TraI

Cited by 90 publications

References 56 publications

Disrupting antibiotic resistance propagation by inhibiting the conjugative DNA relaxase

Disrupting antibiotic resistance propagation by inhibiting the conjugative DNA relaxase

Chapter 12 Using Fluorophore-Labeled Oligonucleotides to Measure Affinities of Protein–DNA Interactions

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

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