Journal: PLoS ONE
Article Title: Mechanistic Insights on the Inhibition of C5 DNA Methyltransferases by Zebularine
Figure Lengend Snippet: Native TBE and SDS-PAGE analysis of the binding and cross-linking between DNMT1 (A and B), DNMT3a/L (C and D) to DNA. M. S ssI was used as control. 5′ [γ 32 P]-labeled duplexes ds-C, ds-Z, ds-F (for M Sss .I and DNMT3a/3L) or mds-C, mds-Z, mds-F (for DNMT1) (0.05 pmol) were incubated with the specified methyltransferase. DNMT1 and DNMT3a/3L complex were incubated in buffer A supplemented by 300 µM of AdoMet; M.SssI was incubated in the buffer supplied by NEB supplemented with 300 µM of AdoMet and 100 µg/mL of bovine serum albumin (BSA). ( A ) DNMT1 was used in excess of once (1X), twice (2X) or 5 times (5X) compared to the duplexes. Incubations were carried out for 4 h at 37°C. Samples were loaded on a 4% native TBE gel. ( B ) mds-X were incubated with DNMT1 at 28 nM final concentration for 16 h at 16°C (lanes 3, 7 and 11) or 16 h at 30°C (lanes 4, 8 and 12). As control ds-C, ds-Z and ds-F (lanes 2, 6 and 10, respectively) were incubated qith 0.5 µL of M.SssI (ca. 0.1 mg/mL) 16 h at 16°C. Samples were loaded on a 10% SDS-PAGE gel. ( C ) DNMT3a/3L complex was used in excess of 10 (10X) to 500 (500X) compared to the duplexes. Incubations were carried out for 1 h at 37°C. Samples were loaded on a 4% native TBE gel. ( D ) ds-X were incubated with DNMT3a/3L complex at 0.75 µM (final concentration) or 0.5 µL of M.SssI (ca. 0.1 mg/mL). Incubations were carried out for 16 h at 30°C (for DNMT3a/3L) or 16°C (for M.SssI). The reaction mixture was loaded onto a 10% denaturing SDS-PAGE. Schematic representation of the labeled single-stranded DNA (––*) or ds ( = *) are indicated on the right of the gels and the circle represents the enzyme bound to labeled single-stranded or double-stranded DNA.
Article Snippet: The reaction mixture was loaded onto a pre-run 4% non-denaturing polyacrylamide gel in TBE buffer and protein-DNA complexes were visualized on a Typhoon (GE Healthcare).
Techniques: SDS Page, Binding Assay, Labeling, Incubation, Concentration Assay