Supplementary Figure 7: Telomerase-associated nuclease removes a primer terminal 8-oxoG.
From: Oxidative guanine base damage regulates human telomerase activity
(a) Telomerase preparations lack contaminating exonuclease activity. 8-oxoG substitutions in the 4R substrate are indicated in red “Go” (oligos 6-8 in Supplementary Table 1). Reactions (20 μl) contained 19.5 pmol primer and 0.5 pmol 32P end-labeled primer for a final total concentration of 1 μM in 1x human telomere buffer. The dNTPs were omitted. Reactions were initiated by adding 6 μl telomerase extract and incubated for 60 min at 30oC, and then run on polyacrylamide denaturing gels. (b) Telomerase reactions were conducted using primers containing three or four TTAGGG repeats, with a 3’ terminal G or 8-oxoG (oligos 3, 4, 6 and 7 Supplementary Table 1). Reactions contained high dNTPs (lane 1) or 2.9 μM dGTP (lanes 2-5) and 0.3 μM 32P-α-dGTP. Products were separated on polyacrylamide denaturing gels. The LC was 32P end-labeled 18-mer oligonucleotide. Bands representing addition (+) or subtraction (-) of telomeric repeats are indicated for 3 repeat (left) and 4 repeat (right) substrate. Product size and corresponding sequence are shown, red = radiolabeled G insertion. (c) Products were quantitated and normalized to the LC, and used to calculate relative activity. Bars represent the mean and s.d. from three independent reactions. Asterisks, ** p < 0.01, *** p < 0.001 base on two-tailed Student’s t test.
