Figure 4.
Effect of methylphosphonate modification on the elongation of chimeric substrates. The standard elongation assay was conducted using partially purified human telomerase from Raji cells and an oligonucleotide as indicated above each lane; black for telomeric DNA and white for nontelomeric DNA, where * indicates the position of a methylphosphonate linkage. Oligonucleotide sequences are listed in Table 1. Products from the standard elongation assay were resolved on a 10% polyacrylamide sequencing gel. All oligonucleotides used in these experiments were subjected to gel purification. (A) Elongation of the primer (G3T2A)2X9 containing methylphosphonate substitutions. Lanes 1-3, (G3T2A)2X9; lanes 4-6, (G3T2A)2X5·X4 primer modified with a methylphosphonate linkage within the 3′ nontelomeric DNA; lanes 7-9, (G3T2A)2·X9 primer modified with a methylphosphonate linkage at the telomeric/nontelomeric DNA boundary. The P<I products generated by the chimeric oligonucleotides are indicated by a bracket at right. Note that the products in the upper region of lane 2 were not RNase A sensitive (compare with lane 3) and did not occur in all experiments. However, the P>I products in lane 8 were sensitive to RNase A treatment (compare with lane 9). Input oligonucleotides were 32P-5′ end-labeled for reference (I; lanes 1, 4, and 7). Samples pretreated with RNase A are designated above by + (lanes 3, 6, and 9). (B) Elongation of the primer (T2AG3)2X12 containing methylphosphonate substitutions. Lanes 1-3, (T2AG3)2; lanes 4-6, (T2AG3)2X12; lanes 7-9, (T2AG3)2X6·X6 primer modified with a methylphosphonate linkage in the 3′ nontelomeric DNA; lanes 10-12, (T2AG3)2·X12 primer modified with a methylphosphonate residue at the telomeric/nontelomeric DNA boundary; lanes 13-15, (T2AG3)2·X12 containing a methylphosphonate residue at the telomeric/nontelomeric DNA boundary and a terminal dideoxy TTP. 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). Sample pretreatment with RNase A is represented above the respective lanes (+; lanes 3, 6, 9, 12, and 15). An arrow to the right indicates the position of the P<I product generated by the chimeric oligonucleotides. RNase A-insensitive products are observed in lanes 5, 6, 8, 9, 11, and 12. RNase A-sensitive P>I products were generated by incubation with (T2AG3)2·X12 (lane 11).