circular ssdna template  (New England Biolabs)

Journal: Methods in enzymology

Article Title: Single-Stranded DNA Curtains for Studying Homologous Recombination

doi: 10.1016/bs.mie.2016.08.005

Figure Lengend Snippet: Different types of DNA curtains. (A) Schematic illustration of a single-tethered DNA curtain made with a linear barrier. (B) Double-tethered DNA curtain where the downstream ends of the DNA are tethered to the exposed anchor points that project above the bilayer. Both formats are compatible with either dsDNA or ssDNA. Adapted with permission from Silverstein, T. D., Gibb, B., & Greene, E. C. (2014). Visualizing protein movement on DNA at the single-molecule level using DNA curtains. DNA Repair (Amst), 20, 94–109.

Article Snippet: Preparation of ssDNA Our ssDNA curtain experiments make use of relative long ssDNA substrates (≥40,000 nucleotides) that are made by rolling circle replication with a biotinylated oligonucleotide primer and a circular ssDNA template ( Gibb et al., 2012 ), as described later. list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 The biotinylated primer is first annealed to a circular M13 DNA template in a 100-μL reaction containing: 40 m M Tris-HCl [pH 8.0], 50 m M NaCl, 10 m M MgCl 2 , 10 μg (89.4 n M ) of M13mp18 (New England Biolabs, Cat. No. N4040S), and 45 n M primer (5′-BIO-TEG-TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT GTA AAA CGA CGG CCA GT).

Techniques:

Journal: Methods in enzymology

Article Title: Single-Stranded DNA Curtains for Studying Homologous Recombination

doi: 10.1016/bs.mie.2016.08.005

Figure Lengend Snippet: RPA-coated ssDNA curtains. (A) Wide-field TIRFM image of a double-tethered ssDNA curtain bound by RPA-eGFP. (B) Kymograph showing what takes place when single-tethered ssDNA molecules are labeled with RPA-eGFP. The ssDNA slowly becomes longer as RPA-eGFP binds and disrupts existing secondary structure. (C) Kymograph showing facilitated dissociation of RPA from the ssDNA when free RPA is injected into the sample chamber. RPA-eGFP is shown in green, and RPA-mCherry is shown in magenta, and the color-coded arrowheads indicate successive injections of each protein. (D) Kymographs of a single-tethered ssDNA showing that the exchange of wild-type RPA-eGFP with wild-type RPA-mCherry does not alter ssDNA length (upper panel), whereas exchange of the RPAt48 mutant, which is defective for ssDNA binding, with wild-type RPA-mCherry coincides with an increase in ssDNA length. Adapted with permission from Deng, S. K., Gibb, B., de Almeida, M. J., Greene, E. C., & Symington, L. S. (2014). RPA antagonizes microhomology-mediated repair of DNA double-strand breaks. Nature Structural and Molecular Biology, 21, 405–412; Gibb, B., Silverstein, T. D., Finkelstein, I. J., & Greene, E. C. (2012). Single-stranded DNA curtains for real-time single-molecule visualization of protein-nucleic acid interactions, Analytical Chemistry, 84, 7607–7612; Gibb, B., Ye, L. F., Gergoudis, S. C., Kwon, Y., Niu, H., Sung, P., et al. (2014). Concentration-dependent exchange of replication protein A on single-stranded DNA revealed by single-molecule imaging. PloS One, 9, e87922; Gibb, B., Ye, L. F., Kwon, Y., Niu, H., Sung, P., & Greene, E. C. (2014). Protein dynamics during presynaptic-complex assembly on individual single-stranded DNA molecules. Nature Structural and Molecular Biology; and Qi, Z., Redding, S., Lee, J. Y., Gibb, B., Kwon, Y., Niu, H., et al. (2015). DNA sequence alignment by microhomology sampling during homologous recombination. Cell, 160, 856–869.

Article Snippet: Preparation of ssDNA Our ssDNA curtain experiments make use of relative long ssDNA substrates (≥40,000 nucleotides) that are made by rolling circle replication with a biotinylated oligonucleotide primer and a circular ssDNA template ( Gibb et al., 2012 ), as described later. list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 The biotinylated primer is first annealed to a circular M13 DNA template in a 100-μL reaction containing: 40 m M Tris-HCl [pH 8.0], 50 m M NaCl, 10 m M MgCl 2 , 10 μg (89.4 n M ) of M13mp18 (New England Biolabs, Cat. No. N4040S), and 45 n M primer (5′-BIO-TEG-TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT GTA AAA CGA CGG CCA GT).

Techniques: Labeling, Injection, Mutagenesis, Binding Assay, Concentration Assay, Imaging, Sequencing, Sampling, Homologous Recombination

Journal: Methods in enzymology

Article Title: Single-Stranded DNA Curtains for Studying Homologous Recombination

doi: 10.1016/bs.mie.2016.08.005

Figure Lengend Snippet: Kymographs showing presynaptic complex assembly reactions. (A) RPA-eGFP-ssDNA curtains were first incubated with 1 μM S. cerevisiae wild-type (unlabeled) Rad51 and 2.5 mM ATP. Binding of Rad51 to the ssDNA is revealed as a rapid loss of RPA-eGFP fluorescence signal. The sample chambers were then flushed (1st chase) with buffer containing 1.0 nM RPA-eGFP and either no nucleotide or 2.5 mM of the indicated nucleotide cofactor, followed by a 30-min incubation. Disassembly of the Rad51-ssDNA presynaptic filaments is revealed by the binding of RPA-eGFP to the exposed ssDNA. The sample chambers were then flushed (2nd chase) with additional buffer containing 0.1 nM RPA-eGFP and no nucleotide cofactor, and incubated for an additional 30-min. (B) Quantitation of the Rad51 filament stability in the presence of various nucleotide cofactors, as indicated. Error bars represent s.d. Adapted with permission from Qi, Z., Redding, S., Lee, J. Y., Gibb, B., Kwon, Y., Niu, H., et al. (2015). DNA sequence alignment by microhomology sampling during homologous recombination. Cell, 160, 856–869.

Article Snippet: Preparation of ssDNA Our ssDNA curtain experiments make use of relative long ssDNA substrates (≥40,000 nucleotides) that are made by rolling circle replication with a biotinylated oligonucleotide primer and a circular ssDNA template ( Gibb et al., 2012 ), as described later. list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 The biotinylated primer is first annealed to a circular M13 DNA template in a 100-μL reaction containing: 40 m M Tris-HCl [pH 8.0], 50 m M NaCl, 10 m M MgCl 2 , 10 μg (89.4 n M ) of M13mp18 (New England Biolabs, Cat. No. N4040S), and 45 n M primer (5′-BIO-TEG-TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT GTA AAA CGA CGG CCA GT).

Techniques: Incubation, Binding Assay, Fluorescence, Quantitation Assay, Sequencing, Sampling, Homologous Recombination