DNA Recognition by F Factor TraI36:  Highly Sequence-Specific Binding of Single-Stranded DNA

Stern, J. C.; Schildbach, Joel F.

doi:10.1021/bi010877q

Cited by 44 publications

(98 citation statements)

References 41 publications

(82 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The changes in fluorescence intensity (top) and anisotropy (bottom) are shown for a 17-base oligonucleotide (5′-TTTGCGTGGGGTGTGTG-3′) 3′-labeled with TAMRA (open circles), 6-FAM (filled triangles), or Cy3 (filled squares). The solid lines depict simultaneous fits to intensity and anisotropy data using SPECTRABIND (Toptygin and Brand, 1995a;Toptygin and Brand, 1995b) as described (Stern and Schildbach, 2001). The linkage between fluorophore and DNA can affect fluorescent properties.…”

Section: Discussionmentioning

confidence: 99%

“…TraI36 titrations into solutions of 22-base (5′-TTTGCGTGGGGTGTGTGCTTTT-3′) singlestranded oligonucleotide 3′-labeled with TAMRA using either a CPG (open circles) or NHS (filled diamonds) linkage are shown. The solid lines depict simultaneous fits to intensity and anisotropy data using SPECTRABIND (Toptygin and Brand, 1995a;Toptygin and Brand, 1995b) as described (Stern and Schildbach, 2001). The proximity of fluorophore to bound protein can affect fluorescent properties.…”

Section: Discussionmentioning

confidence: 99%

“…Intensity (top) or anisotropy (bottom) changes occurring upon titration of TraI36 are shown. The solid lines depict simultaneous fits to intensity and anisotropy data using SPECTRABIND (Toptygin and Brand, 1995a;Toptygin and Brand, 1995b) as described (Stern and Schildbach, 2001). Changing solution conditions can alter fluorescent behavior of probes.…”

Section: Discussionmentioning

confidence: 99%

“…In contrast, ITC experiments can be performed with unmodified DNA oligonucleotides, and EMSA experiments can use oligonucleotides end-labeled with 32 P phosphate, a comparatively minor modification. To check whether the fluorophore is affecting binding, routine characterization of an oligonucleotide should include comparing the measured K D of binding with the measured K I of an unlabeled version of the same oligonucletide measured by competition experiments (Stern and Schildbach, 2001;Titolo et al, 2003b). (Competition assays are discussed later in this review.)…”

Section: Disadvantages Of Fluorescence Measurementsmentioning

confidence: 99%

“…To illustrate this point, we provide curves generated by the binding of TraI36 to single-stranded DNA (ssDNA) oligonucleotides 3′-end-labeled with different fluorophores. TraI36, a 36-kDa N-terminal fragment of the TraI protein of the conjugative bacterial plasmid F factor, binds with sequence-specificity and subnanomolar K D to a single-stranded oligonucleotide containing a plasmid sequence (Stern and Schildbach, 2001). All oligonucleotides were synthesized and HPLC-purified by Integrated DNA Technologies.…”

Section: Designing the Oligonucleotidementioning

confidence: 99%

See 4 more Smart Citations

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

Anderson

Larkin

Guja

et al. 2008

Methods in Enzymology

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Disadvantages Of Fluorescence Measurementsmentioning

confidence: 99%

Section: Designing the Oligonucleotidementioning

confidence: 99%

See 3 more Smart Citations

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

Anderson

Larkin

Guja

et al. 2008

Methods in Enzymology

Self Cite

View full text Add to dashboard Cite

show abstract

Solution structure and small angle scattering analysis of TraI (381–569)

et al. 2012

Self Cite

View full text Add to dashboard Cite

TraI, the F plasmid-encoded nickase, is a 1756 amino acid protein essential for conjugative transfer of plasmid DNA from one bacterium to another. While crystal structures of N- and C-terminal domains of F TraI have been determined, central domains of the protein are structurally unexplored. The central region (between residues 306 and 1520) is known to both bind single-stranded DNA (ssDNA) and unwind DNA through a highly processive helicase activity. Here, we show that the ssDNA binding site is located between residues 381 and 858, and we also present the high-resolution solution structure of the N-terminus of this region (residues 381 to 569). This fragment folds into a four-strand parallel β sheet surrounded by α helices, and resembles the structure of the N-terminus of helicases such as RecD and RecQ despite little sequence similarity. The structure supports the model that F TraI resulted from duplication of a RecD-like domain and subsequent specialization of domains into the more N-terminal ssDNA binding domain and the more C-terminal domain containing helicase motifs. In addition, we provide evidence that the nickase and ssDNA binding domains of TraI are held close together by an 80-residue linker sequence that connects the two domains. These results suggest a possible physical explanation for the apparent negative cooperativity between the nickase and ssDNA binding domain.

show abstract

Relaxases and Plasmid Transfer in Gram-Negative Bacteria

Zechner

Moncalián

Cruz

2017

Current Topics in Microbiology and Immunology

View full text Add to dashboard Cite

All plasmids that spread by conjugative transfer encode a relaxase. That includes plasmids that encode the type IV secretion machinery necessary to mediate cell to cell transfer, as well as mobilizable plasmids that exploit the existence of other plasmids' type IV secretion machinery to enable their own lateral spread. Relaxases perform key functions in plasmid transfer by first binding to their cognate plasmid as part of a multiprotein complex called the relaxosome, which is then specifically recognized by a receptor protein at the opening of the secretion channel. Relaxases catalyze a site- and DNA-strand-specific cleavage reaction on the plasmid then pilot the single strand of plasmid DNA through the membrane-spanning type IV secretion channel as a nucleoprotein complex. In the recipient cell, relaxases help terminate the transfer process efficiently and stabilize the incoming plasmid DNA. Here, we review the well-studied MOB family of relaxases to describe the biochemistry of these versatile enzymes and integrate current knowledge into a mechanistic model of plasmid transfer in Gram-negative bacteria.

show abstract

DNA Recognition by F Factor TraI36: Highly Sequence-Specific Binding of Single-Stranded DNA

Cited by 44 publications

References 41 publications

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

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

Solution structure and small angle scattering analysis of TraI (381–569)

Relaxases and Plasmid Transfer in Gram-Negative Bacteria

Contact Info

Product

Resources

About