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(T4G4T4G4T4G2) 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 (i) 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-32P]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 span the telomerase active and anchor sites. When the single-stranded primers are aligned with the G-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-iododeoxycytidine into the CA strand of the duplex region of telomere analogs. We conclude that the anchor site in the approximately 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 showed that only 60% of the primer remains bound after each repeat addition.
Hammond
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Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215, USA.
Lively
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TN
Cech
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D000818:Animals; D001665:Binding Sites; D001972:Bromodeoxycytidine / Q000031:analogs & derivatives; D003432:Cross-Linking Reagents; D004247:DNA; D017931:DNA Primers; D003841:Deoxycytidine / Q000031:analogs & derivatives; D016812:Euplotes / Q000201:enzymology*; D007065:Idoxuridine; D008957:Models, Genetic; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D019098:Telomerase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D017931:DNA Primers; D003841:Deoxycytidine; D001972:Bromodeoxycytidine; C011288:ibacitabine; D012313:RNA; D004247:DNA; D019098:Telomerase; D007065:Idoxuridine