Journal: Nucleic Acids Research

Article Title: DNA processing by the MOB H family relaxase TraI encoded within the gonococcal genetic island

doi: 10.1093/nar/gkz577

Figure Lengend Snippet: TraI cleaves ssDNA in a sequence specific manner. ( A ) Map of the GGI region containing the oriT. yaf encodes a protein of unknown function and traI the relaxase. ltgX encodes a putative lytic transglycosylase. Arrows mark the promoter regions for the yaf-yaa and yaf-traF transcripts. The 75 nucleotides long oligonucleotide ( oriT -ssDNA) which was used for the cleavage assays is indicated below. The IR sequences and the nic -site are indicated. ( B – D ) Cy3-labeled oligonucleotides were incubated with TraI for 1 h at 25°C and separated on denaturing polyacrylamide-urea gels. Representative gels from at least 3 experiments are shown. (B) 500 nM [Cy3]- oriT -ssDNA was incubated with 10 μM TraI(28–427) in the presence of increasing concentrations of Mn 2+ or Co 2+ . Marker lanes show labeled polyT oligonucleotides with indicated lengths. oriT containing oligonucleotides migrated slower than expected based on their size, most likely because the hairpin structure was not completely denatured. (C) 500 nM [Cy3]- oriT -ssDNA was incubated with 10 μM TraI(28–427) in the absence (–) and presence (+) of 5 mM Mn 2+ and separated on a high-resolution polyacrylamide-urea gel. Marker lane shows oligonucleotides representing 16, 17 and 18 nucleotides of the 5′ end of the oriT -ssDNA. (D) 500 nM [Cy3]- oriT -ssDNA (5′Cy3) and oriT -ssDNA-[Cy3] (3′Cy3) were incubated with 10 μM TraI(28–427), TraI(42–850) or TraI(674–850) in the absence (–) and presence (+) of 5 mM Mn 2+ . Marker lanes show labeled polyT oligonucleotides with indicated lengths. (E) 2 μM (squares) and 5 μM (triangles) TraI(28–427) (black symbols and line) or TraI(42–850) (gray symbols and line) were incubated with 10 μM [Cy3]- oriT -ssDNA in the presence of 5 mM Mn 2+ for up to 180 min. Samples were separated on denaturing polyacrylamide-urea gels and the relative cleavage of oriT -ssDNA was determined. Standard deviations derived from three to four independent experiments are shown. ( F ) 25 μM TraI(28–427) was incubated with 2 μM unlabeled oriT -ssDNA in the absence (–) and in the presence (+) of 5 mM Mn 2+ for 2 h. TraI was heat-inactivated and subsequently incubated with T4-polynucleotide kinase (PNK) in the presence of γ-[ 32 P]-ATP for 1 h followed by separation on denaturing polyacrylamide-urea gels. As size controls, oligonucleotides representing the uncleaved oriT -ssDNA , the 5′ cleavage product and the 3′ cleavage product were subjected to 5′ labeling by PNK. A representative gel from 4 experiments is shown. In the schematic on the right, the different grey tones of the oligonucleotides correspond to the colors of the arrows.

Article Snippet: 20 U T4-PNK, 10× PNK buffer and 0.125 μCi γ- 32 P-ATP (Hartmann Analytic) were added.

Techniques: Sequencing, Labeling, Incubation, Marker, Derivative Assay