FIG. 6.
The effect of noncognate single-strand binding proteins on telomerase activity and of POT1 on terminal transferase activity. (A) Direct telomerase assays with single-strand binding protein bound primers. Primer GGGTTA3 was incubated with dilution buffer (lanes 1 and 5), E. coli SSB (50, 100, or 500 nM; lanes 2 to 4), or T4 gene 32 protein (0.1, 1, or 10 μM; lanes 6 to 8), following which the extension reactions were initiated by the addition of in vitro-transcribed and -translated telomerase. Lower panel, B-10mer loading control (LC). (B) Terminal transferase activity assay. Primer GGGTTA3 was incubated with dilution buffer (lanes 1), POT1 (124, 247, or 425 nM; lanes 2 to 4), ΔOB (124, 247, or 425 nM; lanes 5 to 7), or purified vector control lysate (dilutions equivalent to those of POT1; lanes 8 to 10) Extension reactions were initiated by the addition of terminal transferase (0.4 U), and incubation continued for 1 h. Products were separated on a 10% sequencing gel. +1 indicates the position of the first incorporated nucleotide.