FIG. 5.

Distinct hTR regions are essential for DNA synthesis and repeat addition processivity. (A and B) Schematics of the predicted secondary structures of the hTR CR4-CR5 domain (A) and pseudoknot P3 base pairing region (B) (17, 20, 43). (A) The starting positions of mutations analyzed in this study are indicated. (B) The starting positions of the hTR170, hTR180, and hTR190 10-nucleotide substitutions are numbered, and the location of the GC107-108AG (DKC) mutation is underlined. P3 residues implicated in hTR dimerization are indicated by uppercase letters (40). CR2 and CR3 are boxed. (C and D) The elongation products of telomerases reconstituted with wild-type (WT) hTERT and the indicated CR4-CR5 (C) or pseudoknot (D) substitution mutants were examined using the direct primer extension assay. Repeat addition processivity (R.A.P.) and first telomere repeat DNA synthesis (DNA synthesis) values were expressed as percentages of the corresponding values for wild-type enzyme and are indicated below the gels. n/a, not applicable. (E) Interaction of wild-type (WT) hTR and P6.1 helix mutants with wild-type hTERT and RID2 (amino acids 300 to 617). The WT hTERT proteinsignal in lane 4 appears stronger than the WT hTERT protein signals in lanes 7 and 10 due to comigration of the efficiently coprecipitated WT hTR with hTERT in SDS-12% polyacrylamide gels. One microliter of a 1:500 dilution of an input RRL reaction mixture containing the appropriate ³²P-labeled hTR variant was loaded in input lanes. PI, preimmune serum. (F) Quantification of in vitro hTR dimerization. At least three independent experiments were performed for each hTR. Mean dimerization values that differed significantly from the value for wild-type (WT) hTR in a Student's t test are indicated by one asterisk (P < 0.05) or two asterisks (P < 0.01). (G) Representative hTR dimerization results. hTRs were incubated on ice (−) or at 37°C (+) for 2 h prior to electrophoresis on 5% nondenaturing polyacrylamide gels. The positions of monomers (one asterisk) and dimers (two asterisks) are indicated to the right of the gel.