Functional Organization of Repeat Addition Processivity and DNA Synthesis Determinants in the Human Telomerase Multimer

Abstract

Human telomerase is a multimer containing two human telomerase RNAs (hTRs) and most likely two human telomerase reverse transcriptases (hTERTs). Telomerase synthesizes multiple telomeric repeats using a unique repeat addition form of processivity. We investigated hTR and hTERT sequences that were essential for DNA synthesis and processivity using a direct primer extension telomerase assay. We found that hTERT consists of two physically separable functional domains, a polymerase domain containing RNA interaction domain 2 (RID2), reverse transcriptase (RT), and C-terminal sequences, and a major accessory domain, RNA interaction domain 1 (RID1). RID2 mutants defective in high-affinity hTR interactions and an RT catalytic mutant exhibited comparable DNA synthesis defects. The RID2-interacting hTR P6.1 helix was also essential for DNA synthesis. RID1 interacted with the hTR pseudoknot-template domain and hTERT's RT motifs and putative thumb and was essential for processivity, but not DNA synthesis. The hTR pseudoknot was essential for processivity, but not DNA synthesis, and processivity was reduced or abolished in dimerization-defective pseudoknot mutants. trans-acting hTERTs and hTRs complemented the processivity defects of RID1 and pseudoknot mutants, respectively. These data provide novel insight into the catalytic organization of the human telomerase complex and suggest that repeat addition processivity is one of the major catalytic properties conferred by telomerase multimerization.

We thank L. Harrington for donating the pCR3.1hTERT plasmid used to generate Flag-hTERT constructs, R. Lin for the Flag-tagged NKKβ construct, and M.-C. Boulanger and S. Richard for donating the DART-1 construct. We thank M. A. Cerone, M. Götte, S. Huard, M. Taboski, and R. J. Ward for comments on the manuscript; J. Demers, S. Dupuis, S. Huard, Y. Sun, and M. Taboski for providing technical assistance or constructs; and C. Lalonde for help in preparing the figures.

This work was supported by CIHR grant MOP-14026 to C.A. T.J.M. is the recipient of a CIHR doctoral research scholarship. C.A. was supported by Boehringer Ingelheim (Canada) Young Investigator and FRSQ Chercheur-Boursier awards.