Journal: Nucleic Acids Research

Article Title: Defining characteristics of Tn 5 Transposase non-specific DNA binding

doi: 10.1093/nar/gkl179

Figure Lengend Snippet: Non-specific DNA binding extends the lifetime of Tnp. ( A ) A schematic of reactions to determine Tnp half-life in the presence of non-specific DNA is shown. Tnp was incubated in the absence of non-specific DNA (control) or in the presence of linear or supercoiled pUC19. Aliquots were removed at various times and a ES containing oligonucleotide was added to each time point. The aliquots were then incubated for an additional 90 min during which active Tnp could form a PEC. Tnp is represented as a gray oval, linear and supercoiled non-specific DNA are labeled. The ES containing oligonucleotide is shown as two parallel lines containing a gray box, the *'s represent the fluorescent label at each 5′ end. Inactive Tnp is marked with an ‘X.’ The PEC contains two ES containing oligonucleotides and two molecules of Tnp. ( B ) The lifetime of Tnp in the absence of non-specific DNA (control) was assessed. PECs were separated from unbound oligonucleotide using polyacrylamide gel electrophoresis. The PECs and unbound DNA are labeled as in (A). This experiment was performed twice and the mean percentage of DNA in PECs was determined for each time point. These mean percentages were plotted versus time and the data were fit to a one-phase exponential equation. The error bars associated with each point show the standard error. ( C ) The lifetime of Tnp in the presence of linear pUC19 was assessed. This experiment was performed as in (B) except the time course was extended to 11.9 h. ( D ) The lifetime of Tnp in the presence of supercoiled pUC19 was assessed. This experiment was performed as in (B) except the time course was extended to 24.9 h. ( E ) The lifetime of Tnp is quantitatively expressed as the half-life, or the time at which Tnp activity is half maximal. The half-life of Tnp under each of the previous conditions was determined from the exponential fit of each dataset (see Materials and Methods). This table shows the half-life of Tnp under each reaction condition and the fold extension in half-life due to the presence of non-specific DNA.

Article Snippet: These included DNA binding buffer, DNA binding buffer plus 1 mg/ml Herring Sperm DNA (Promega) and DNA binding buffer plus 1 μM 60 bp double stranded oligonucleotide having the Tnp ES [sequence described in , Integrated DNA Technologies].

Techniques: Binding Assay, Incubation, Control, Labeling, Polyacrylamide Gel Electrophoresis, Activity Assay

Journal: Nucleic Acids Research

Article Title: Defining characteristics of Tn 5 Transposase non-specific DNA binding

doi: 10.1093/nar/gkl179

Figure Lengend Snippet: Tnp can dissociate from λ-DNA in the absence of the Tnp ES. ( A ) To prepare DNA for single molecule micromanipulation experiments, digoxigenin-labeled oligonucleotide was ligated to the λ-DNA molecules using the cosR site and biotin was added using the cosL site. A magnetic strepavidin coated bead was attached to the λ-DNA via interaction with the biotin and the λ-DNA molecules were then coupled to an anti-digoxigenin coated glass slide to create the final substrate. ( B ) To investigate the stability of a linear non-specific DNA–Tnp complex, single molecule experiments were performed. Reaction components are defined as in . For each experiment, a single λ-DNA molecule was isolated and allowed to fully condense with Tnp at 0.04 pN. The force was increased to 1.0 pN and then three individual experiments were performed (represented by arrows). First, the affect of non-specific DNA binding buffer alone was investigated. Next, the affect of non-specific DNA on the Tnp–λ-DNA complex was assessed. Finally, the Tnp–λ-DNA complex was challenged with a double stranded oligonucleotide having the transposon ES. At least five extension measurements were made following the 1 h incubation at 1 pN. The average λ-DNA extensions are shown as gray bars above schematics of Tnp–λ-DNA binding behavior under each condition. The error bars represent one standard deviation from the mean.

Article Snippet: These included DNA binding buffer, DNA binding buffer plus 1 mg/ml Herring Sperm DNA (Promega) and DNA binding buffer plus 1 μM 60 bp double stranded oligonucleotide having the Tnp ES [sequence described in , Integrated DNA Technologies].

Techniques: Micromanipulation, Labeling, Isolation, Binding Assay, Incubation, Standard Deviation