Figure 5.

Sequential binding of two active sites to substrates suggests a sequential kinetic model for dimeric enzymes. A, schematic representation of tethered telomerase being tested for whether two active sites are saturated by substrates at the same time. B, ddTRAP assay on samples obtained from experiments as designed in A. Telomerase extension products were collected, and equal amounts were used for the ddTRAP assay. Error bars, S.D. (n = 3). C, ddTRAP assay of tethered telomerase performed at varying times for extension 1 only. Blue, gray, and black bars represent 30, 90, and 120 min, respectively. Extensions 2 and 3 were all performed for 2 h. Ctl** samples were time-delayed samples that were left at room temperature for 5 h before starting the extension reaction for a duration of 30, 90, or 120 min. Error bars, S.D. (n = 3). Enzyme aliquots with cell equivalency of 250 were used for B and C.