THE ANCHOR SITE OF TELOMERASE FROM EUPLOTES AEDICULATUS REVEALED BY PHOTO-CROSS-LINKING TO SINGLE-STRANDED 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 be en observed to add repeats to a DNA oligonucleotide primer in a proces sive manner, leading to the postulation of a DNA anchor site separate from the catalytic site of the enzyme. We have substituted photoreacti ve 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 t he 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 D NA primer and ii) a protein moiety of approximately 130 kDa and (ii) U 51-U52 of the telomerase RNA. The cross-linked primer could be extende d by telomerase in the presence of [alpha-P-32]dGTP, thus indicating t hat the 3' end was bound in the enzyme active site, The locations of t he cross-links within the single-stranded primers were 20 to 22 nucleo tides upstream of the 3' end, providing a measure of the length of DNA required to spun the telomerase active and anchor sites, When the sin gle-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 obtaine d by substitution of 5-iododeoxyeutidine into the CA strand of the dup lex region of telomerase analogs, We conclude that the anchor site in the similar to 130-kDa protein can bind duplex as well as single-stran ded DNA which may be critical for its function at chromosome ends, Qua ntitation of the processivity with single-stranded DNA primers and dou ble-stranded primers with 3' tails shelved that only 60% of the primer remains bound after each repeat addition.