Figure 3. Quantification of the TERT monomer vs multimer content in purified samples based on TERT-labeling efficiency.
(A) SDS-PAGE analysis of the kinetics of labeling F-purified 293T- or RRL-reconstituted MCP-TERT in reactions with CoA-Cy5 or CoA-Cy3 and SFP synthase. Lines within the panel indicate separate sets of gel lanes. Quantification of labeling intensity was normalized to labeling at the 4-hr time point after subtraction of background. (B) Cy5 labeling relative to TERT amount analyzed for telomerase reconstituted and purified as indicated. TERT was detected by TERT immunoblot. Values are the average of triplicate experimental replicates. (C) Validation of equivalent labeling using CoA-dye or CoA-biotin by sequential labeling of F-purified, RRL-expressed MCP-TERT with SFP synthase. Initial TERT labeling using CoA-Cy3 or CoA-biotin competes for subsequent TERT labeling by the other CoA derivative. The biotin label on MCP-TERT was detected by biotin antibody immunoblot. (D) Left: schematic of the biotinylated TERT depletion procedure. Right: quantification of ACP- and/or MCP-TERT remaining after streptavidin agarose depletion, following reconstitution (RRL unless indicated otherwise), F-purification and labeling using CoA-biotin and ACP (A) or SFP (S) synthase. RRL-expressed TERT was 35S-methionine labeled and 293T-expressed TERT was detected by FLAG antibody immunoblot. Samples labeled in reactions lacking CoA-biotin (not Biotin +) were labeled with CoA and those not applied to streptavidin agarose (not Streptavidin depletion +) were mock-depleted on Myc antibody agarose. Lines within the panel indicate separate sets of gel lanes run in parallel. Percentage unbound was calculated as unbound signal normalized to unbound signal of the control depletion. Values are the average of triplicate experimental replicates. (E) Activity of the unbound fraction after streptavidin agarose depletion of biotinylated telomerase labeling using CoA-biotin and SFP synthase, under native binding conditions. Telomerase activity was assayed in reactions with dATP, dGTP, dTTP, and α-32P dGTP, followed by denaturing gel electrophoresis; number of 6-nucleotide repeats added to product DNA is indicated. Samples not depleted with streptavidin agarose were mock-depleted on Myc antibody agarose. Lines within the panel indicate separate sets of gel lanes run in parallel. Percentage unbound was normalized to unbound after control depletion. Values are the average of triplicate experimental replicates. (F) Left: schematic of the biotinylated TERT depletion procedure and unbound fraction analysis. Right: quantification of total TERT and biotinylated MCP-TERT in the unbound fraction of 293T-reconstituted, F-purified telomerase, following labeling using CoA-biotin or CoA and depletion by streptavidin agarose or mock-depletion on Myc antibody agarose. MCP-TERT and the biotin label on MCP-TERT were detected by immunoblot. Values are the average of triplicate experimental replicates. (G) Illustration of labeling efficiency determination by comparison of the percent unbound total MCP-TERT and unbound biotinylated MCP-TERT. (H) Calculated percentage of DNA-bound TERT monomer complexes according to fraction TERT subunit co-localization (percentages indicated), assuming the TERT-labeling efficiency measured value (82%, blue line; bar graph at right), lower bound (51%, green line; Low L numbers at right), or upper bound (100%, purple line; High L numbers at right). Vertical dashed lines are the observed fraction of two-color co-localization (from Figure 2A). (I) Calculated percentage of DNA-bound TERT monomer complexes according to fraction of one-step photobleaching (percentages indicated), assuming the TERT-labeling efficiency measured value (82%, blue line; bar graph at right), lower bound (51%, green line; High L numbers at right), or upper bound (100%, purple line; Low L numbers at right). Vertical dashed lines are the observed fraction of one-step photobleaching (from Figure 2B).