Marseille Cancer Research Center CRCM, U1068 Inserm, UMR7258 CNRS, Aix-Marseille Univ, Institut Paoli-Calmettes, Marseille, France.
EMBO J. 2012 Apr 18;31(8):2034-46. doi: 10.1038/emboj.2012.40. Epub 2012 Feb 21.
In Saccharomyces cerevisiae, the telomerase complex binds to chromosome ends and is activated in late S-phase through a process coupled to the progression of the replication fork. Here, we show that the single-stranded DNA-binding protein RPA (replication protein A) binds to the two daughter telomeres during telomere replication but only its binding to the leading-strand telomere depends on the Mre11/Rad50/Xrs2 (MRX) complex. We further demonstrate that RPA specifically co-precipitates with yKu, Cdc13 and telomerase. The interaction of RPA with telomerase appears to be mediated by both yKu and the telomerase subunit Est1. Moreover, a mutation in Rfa1 that affects both the interaction with yKu and telomerase reduces the dramatic increase in telomere length of a rif1Δ, rif2Δ double mutant. Finally, we show that the RPA/telomerase association and function are conserved in Schizosaccharomyces pombe. Our results indicate that in both yeasts, RPA directly facilitates telomerase activity at chromosome ends.
在酿酒酵母中，端粒酶复合物与染色体末端结合，并通过与复制叉进展偶联的过程在晚期 S 期被激活。在这里，我们表明，单链 DNA 结合蛋白 RPA（复制蛋白 A）在端粒复制过程中结合到两个子端粒上，但只有其与前导链端粒的结合依赖于 Mre11/Rad50/Xrs2（MRX）复合物。我们进一步证明，RPA 特异性地与 yKu、Cdc13 和端粒酶共沉淀。RPA 与端粒酶的相互作用似乎是由 yKu 和端粒酶亚基 Est1 共同介导的。此外，Rfa1 中的一个突变，同时影响与 yKu 和端粒酶的相互作用，会降低 rif1Δ、rif2Δ 双突变体中端粒长度的显著增加。最后，我们表明 RPA/端粒酶的关联和功能在裂殖酵母中是保守的。我们的结果表明，在两种酵母中，RPA 直接促进染色体末端的端粒酶活性。
