Interactions between Telomerase and Primase Physically Link the Telomere and Chromosome Replication Machinery

Abstract

In the ciliate Euplotes crassus, millions of new telomeres are synthesized by telomerase and polymerase α-primase during macronuclear development in mated cells. Concomitant with de novo telomere formation, telomerase assembles into higher-order complexes of 550 kDa, 1,600 kDa, and 5 MDa. We show here that telomerase is physically associated with the lagging-strand replication machinery in these complexes. Antibodies against DNA primase precipitated telomerase activity from all three complexes from mated cells but not the 280-kDa telomerase complex from vegetatively growing cells. Moreover, when telomerase was affinity purified, primase copurified with enzyme from mated cells but not with the 280-kDa vegetative complex. Thus, the association of telomerase and primase is developmentally regulated. Intriguingly, PCNA (proliferating cell nuclear antigen) was also found in the 5-MDa complex from mated cells. We therefore speculate that this complex is a complete telomere synthesis machine, while the smaller complexes are assembly intermediates. The physical association of telomerase and primase explains the coordinate regulation of telomeric G- and C-strand synthesis and the efficiency of telomere addition in E. crassus.

We thank Larry Klobutcher for the kind gifts of the LEMAC library and the H3 antibody and Leigh Anne Hurley for generating the A1 and A2 Euplotes strains. We thank Chad Price for constant support and help with DNA sequence analysis.

This work was supported by National Institutes of Health grants RO1 GM41803 to C.M.P. and RO1 GM49157 to D.E.S.