Figure 1. Suv39h1-CD can bind nucleic acids.

(A) Schematic of full-length Suv39h1, GST-fused Suv39h1-CD (39–105), and S. pombe Chp1-CD (20–75). (B) The recombinant GST-fused proteins used in (C); these proteins were visualized by CBB staining. (C) An EMSA using GST-fused proteins. 8 μM of GST or GST-fusion was used in one assay. Fluorescein-labeled 130-nt major satellite ssRNA was used as a probe. (D and E) Titration EMSAs using GST-Suv39h1-CD (0–8 μM with 0.5-fold dilutions) incubated with (D) 130-nt ssRNA or (E) 130 bp dsDNA. DNA probe was detected as doublet bands in gels, because the number of the fluorescent dye, which was conjugated at the single or both 5’-ends, could slightly affect the migration. (F) The binding isotherm of Suv39h1-CD to ssRNA and dsDNA; plots were calculated from the unbound fractions. (G) The dissociation constants measured by titration ssRNA and dsDNA EMSA experiments (D and E).

DOI: http://dx.doi.org/10.7554/eLife.25317.003

Figure 1—figure supplement 1. Characteristics of Suv39h1-full-length and -CD’s RNA binding.

(A), (B), (D), (E), and G–I) EMSAs were conducted with GST-fused Suv39h1-CD (39–105) and the following probes: (A) 130-nt alfa satellite ssRNA, (B) 130-nt ACTB mRNA, (D) 74-nt major satellite ssRNA, (E) 189-nt major satellite ssRNA, (G) 60-nt (UUAGGG)x10 ssRNA, (H) 74-nt major satellite DNA/RNA hybrid, or (I) 130-nt major satellite dsRNA. (C and F) The binding isotherms of Suv39h1-CD to various ssRNA probes. (J) MBP-fusion proteins used in (K–N) were resolved by SDS-PAGE and visualized by Coomassie staining. (K–N) EMSAs were conducted with MBP (K), MBP-fused Suv39h1-CD (39-105) (L), MBP-fused full-length Suv39h1-WT (M) or 4A (N) using 130-nt major satellite ssRNA as the probe. (O) The binding isotherms of MBP-fused Suv39h1 proteins to 130-nt major satellite ssRNA.

DOI: http://dx.doi.org/10.7554/eLife.25317.004