The anchor site of telomerase from Euplotes aediculatus revealed by photo-cross-linking to single- and double-stranded DNA primers

Telomerase is a ribonucleoprotein enzyme that adds telomeric sequence repeats to the ends of linear chromosomes, In vitro, telomerase has been observed to add repeats to a DNA oligonucleotide primer in a processive manner, leading to the postulation of a DNA anchor site separate from the catalytic site of the enzyme. We have substituted photoreactive 5-iododeoxypyrimidines into the DNA oligonucleotide primer d(T(4)G(4)T(4)G(4)T(4)G(2)) and, upon irradiation, obtained cross-links with the anchor site of telomerase from Euplotes aediculatus nuclear extract, No cross-linking occurred with a primer having the same 5' end and a nontelomeric 3' end, These cross-links were shown to be between the DNA primer and ii) a protein moiety of approximately 130 kDa and (ii) U51-U52 of the telomerase RNA. The cross-linked primer could be extended by telomerase in the presence of [alpha-P-32]dGTP, thus indicating that the 3' end was bound in the enzyme active site, The locations of the cross-links within the single-stranded primers were 20 to 22 nucleotides upstream of the 3' end, providing a measure of the length of DNA required to spun the telomerase active and anchor sites, When the single-stranded primers are aligned with the C-rich strand of a Euplotes telomere, the cross-linked nucleotides correspond to the duplex region, Consistent with this finding, a cross-link to telomerase was obtained by substitution of 5-iododeoxyeutidine into the CA strand of the duplex region of telomerase analogs, We conclude that the anchor site in the similar to 130-kDa protein can bind duplex as well as single-stranded DNA which may be critical for its function at chromosome ends, Quantitation of the processivity with single-stranded DNA primers and double-stranded primers with 3' tails shelved that only 60% of the primer remains bound after each repeat addition.