puc18 dna  (Addgene inc)

Journal: Nucleic Acids Research

Article Title: Mlh1–Pms1 couples ATP-driven DNA compaction with nick-dependent endonuclease activation

doi: 10.1093/nar/gkaf1252

Figure Lengend Snippet: Non-B-form regions disrupt Mlh1–Pms1 activities. ( A ) Dinucleotide repeats were inserted into a 2.7 kb pUC18 plasmid to assess impact on Mlh1–Pms1 activities. Oligonucleotides used to construct the inserts are given in . ( B–D ) Electrophoretic mobility shift assays on supercoiled DNA substrates containing dinucleotide repeat regions. Where indicated, Mlh1–Pms1 concentrations are 25, 50, 100, 200, 300, and 400 nM. Binding reactions were incubated at room temperature for 10 min. Experiments are performed in triplicate and representative gels are included. ( E–G ) DNA binding to linear substrates containing a dinucleotide repeat sequence linearized by BsaI-Hfv2, which positions the dinucleotide repeat sequence in the center of the plasmid. For panels (E) and (F) Mlh1–Pms1 was included at 50, 100, 200, 300, and 400 nM. For panel (G), Mlh1–Pms1 was included at 25, 50, 100, 200, 300, and 400 nM. Experiments are performed in triplicate and representative gels are included. ( H ) Endonuclease assays on substrates containing either no repeat sequence, or a (AT) 21 or (GC) 21 repeat sequence. Mlh1–Pms1 was titrated at 10, 25, 50, 100, 150, and 200 nM. The average proportion of supercoiled DNA converted to nicked circular product from three replicates. Error bars are the standard deviation between replicates. Data were fit to a sigmoidal function describing cooperative activity. Representative images are in .

Article Snippet: The topoisomerase was then inactivated by incubating at 80°C for 20 min. Plasmid substrates containing a non-B-form, nucleotide repeat segment were generated by digesting 3.36 pmol of pUC18 with EcoRI (NEB) and BamHI (NEB).

Techniques: Plasmid Preparation, Construct, Electrophoretic Mobility Shift Assay, Binding Assay, Incubation, Sequencing, Standard Deviation, Activity Assay