| Abstract: | A number of published studies indicate that telomerase may interact with oligonucleotide primers in a bipartite manner, with the 3'-end of the primer positioned at the catalytic site of the enzyme and a more 5' region of the primer binding to a second or 'anchor' site of the enzyme. We systematically investigated the effects of mutations in the DNA primer on overall binding and polymerization by yeast telomerase. Our studies indicate that there is sequence-specific interaction between telomerase and a substantial region of the DNA primer. Mutations in the 3'-most positions of the primer reduced polymerization, yet had little effect on overall binding affinity. In contrast, mutations around the -20 position reduced binding affinity but had no effect on polymerization. Most strikingly, mutations centered around the -12 position of the DNA primer reduced overall binding affinity but dramatically enhanced primer extension, as well as primer cleavage. This finding suggests that reduced interaction with the -12 region of the DNA primer can facilitate a step in the catalytic region of yeast telomerase that leads to greater polymerization. A tripartite model of interaction between primer and telomerase is proposed to account for the distinct effects of mutations in different regions of the DNA primer. |
