Figure 1.

ZBTB10 preferably binds the TTGGGG variant repeat sequence. (A) Binding of stably expressed ZBTB10-GFP from HEK293 and U2OS cells to telomeric and telomeric variant repeats. Biotinylated concatenated DNA oligonucleotides were immobilized on streptavidin magnetic beads and bound proteins were analyzed by Western blot. A scrambled GTGAGT repeat sequence was used as control bait. (B) DNA pulldowns with extracts containing recombinantly expressed His₆-ZBTB10 to telomeric and telomeric variant repeats. (C) Schematic representation of constructed ZBTB10 deletion variants together with their respective length in amino acids (aa). (D) Binding of ZBTB10-GFP variants in DNA pulldown experiments stably expressed in U2OS cells. (E) DNA pulldowns of ZBTB10 zinc finger mutants zinc finger 1 (C551A) and zinc finger 2 (C579A) stably expressed in U2OS cells. (F) Fluorescence polarization assays of the minimal ZBTB10 construct consisting of both zinc fingers (ZBTB10-2Znf) and the longer construct containing both C2H2 fingers and the C-terminus (ZBTB10-2Znf-C). Binding affinities to the telomeric variant repeat TTGGGG (black circle), the telomeric repeat TTAGGG (blue square) and the predicted binding motif GCGGGG (green diamond) were determined.