Hammond P W, Lively T N, Cech T R
Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215, USA.
Mol Cell Biol. 1997 Jan;17(1):296-308. doi: 10.1128/MCB.17.1.296.
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.
端粒酶是一种核糖核蛋白酶，可将端粒序列重复添加到线性染色体的末端。在体外，已观察到端粒酶以连续的方式将重复序列添加到DNA寡核苷酸引物上，从而推测存在一个与酶催化位点分开的DNA锚定位点。我们已将光反应性5-碘脱氧嘧啶替代到DNA寡核苷酸引物d(T4G4T4G4T4G2)中，并在照射后，获得了与来自嗜热四膜虫核提取物的端粒酶锚定位点的交联。对于具有相同5'端和非端粒3'端的引物，未发生交联。这些交联显示存在于DNA引物与(i) 一个约130 kDa的蛋白质部分以及(ii) 端粒酶RNA的U51-U52之间。在[α-32P]dGTP存在下，交联的引物可被端粒酶延伸，因此表明3'端结合在酶活性位点中。单链引物内交联的位置在3'端上游20至22个核苷酸处，这提供了跨越端粒酶活性位点和锚定位点所需的DNA长度的量度。当单链引物与嗜热四膜虫端粒的富含G的链对齐时，交联的核苷酸对应于双链区域。与此发现一致，通过将5-碘脱氧胞苷替代到端粒类似物双链区域的CA链中，获得了与端粒酶的交联。我们得出结论，约130 kDa蛋白质中的锚定位点可结合双链以及单链DNA，这可能对其在染色体末端的功能至关重要。对单链DNA引物和具有3'尾巴的双链引物的持续合成能力的定量显示，每次添加重复序列后，仅60%的引物仍保持结合。
