Supplementary Figure 2: Recombinant proteins used in this study and their binding to telomeric dsDNA.

(a) Structural domains of the recombinant proteins used in strand invasion and EMSA assays. GST, glutathione S-transferase; B, basic; A, acidic; TRFH, TRF homodimerization; M, Myb; RH1HBD, RNA:DNA hybrid binding domain of human RNaseH1. Black dots indicate single amino acid substitutions within the TRFH domain of the Topless mutant. (b) 200 ng of recombinant proteins were size-fractionated by SDS-PAGE and stained with EZBlue reagent. Molecular weights of a size marker (m) are on the left in kDa. (c) The indicated proteins (80 nM) were incubated with G-rich RNA or DNA oligonucleotides (10 nM) in the same conditions as for invasion assays, followed by electrophoresis in a denaturing polyacrylamide gel and radioactive signal detection. Note that incubation with proteins did not affect the signal associated to the oligonucleotides, when compared to samples incubated in absence of proteins, thus excluding DNase, RNase and phosphatase contaminations. (d) EMSAs with a radiolabeled dsDNA fragment containing 24 TTAGGG repeats (0.26 nM) and increasing amounts of the indicated recombinant proteins. (e) EMSAs with a radiolabeled dsDNA fragment comprising a ~0.8 kb long (TTAGGG)n array (0.03 nM) and increasing amounts of the indicated recombinant proteins. Bound probes were quantified and graphed as fraction of the total signal within each lane. Values are means ± SD (n = 3 independent experiments). (f) EMSAs as in d using GST fused to A, B or A and B (AB) domains. *, wells.