Figure 5.
Telomerase elongation products generated by oligonucleotides containing 3′ sequence that is partially complementary to the telomerase RNA template. (A) Standard elongation assay was conducted using partially purified human telomerase from Raji cells and the oligonucleotide indicated above each lane: black for telomeric DNA, white for nontelomeric DNA, and striped for G-rich DNA. Refer to Table 1 for oligonucleotide sequences. Standard elongation assay products were resolved on a 10% acrylamide sequencing gel. The 32P-5′ end-labeled input oligonucleotides are shown to the left of their respective telomerase elongation reactions (I; lanes 1, 4, 7, 10, and 13). Those samples pretreated with RNase A are indicated above the corresponding lane (+; lanes 3, 6, 9, 12, and 15). RNase A-insensitive products are indicated by an asterisk alongside the gel (lanes 5 and 6). (B) Schematic for the direct elongation of -3/-25 and the proposed utilization of TA(G3T2A)2-3/-11 by human telomerase. The -3/-25 primer is composed of a G-rich 5′ region (denoted -12/-25) and a 3′ region capable of limited pairing with the telomerase RNA template (denoted -3/-11) (Morin, 1991). As previously shown, the -3/-25 primer can be directly extended by human telomerase (Morin, 1991). Potential base pairing with the telomerase RNA template is indicated above the primer sequence and the newly synthesized telomeric DNA is underlined. Both -3/-25 and TA(G3T2A)2-3/-11 possess the same 3′ sequence (designated -3/-11). Therefore, TA(G3T2A)2-3/-11 may participate in 3′ primer-template base-pairing interactions as with -3/-25. The P>I extension products generated by TA(G3T2A)2-3/-11 may result from the direct extension of this oligonucleotide by telomerase. The telomeric sequence at the 5′ end of TA(G3T2A)2-3/-11 may promote cleavage and subsequent extension by telomerase, consistent with the P<I products observed with this primer. The 5′ product of cleavage at the telomeric/nontelomeric boundary in TA(G3T2A)2-3/-11 is shown (although cleavage may also occur 5′ of the boundary).