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首页> 外文期刊>Journal of Molecular Biology >A new model for Schizosaccharomyces pombe telomere recognition: the telomeric single-stranded DNA-binding activity of Pot11-389.
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A new model for Schizosaccharomyces pombe telomere recognition: the telomeric single-stranded DNA-binding activity of Pot11-389.

机译：粟酒裂殖酵母端粒识别的新模型：Pot11-389的端粒单链DNA结合活性。

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摘要

The protection of telomeres 1 (Pot1) proteins specifically recognize the single-stranded 3' end of the telomere, an activity essential for sustained cellular viability and proliferation. The current model for the telomeric single-stranded DNA (ssDNA) binding activity of Schizosaccharomyces pombe Pot1 is based on a 20 kDa fragment, Pot1pN. Recent biochemical studies suggest that SpPot1 contains a larger ssDNA-binding domain and we have identified a novel ssDNA-binding domain similar in size to the human Pot1 domain. This domain, Pot1(1-389), binds extremely tightly to an oligonucleotide consisting of two conserved hexameric S. pombe telomere repeats, d(GGTTACGGTTAC), with an affinity approximately 4000-fold tighter than Pot1pN binds its cognate ssDNA. The Pot1(1-389)/ssDNA complex exhibits a half-life of 53 min, consistent with that estimated for full-length SpPot1 and significantly longer than that of Pot1pN. Single nucleotide substitutions reveal that, in contrast to Pot1pN, tandem trinucleotide repeats (GTT) within d(GGTTACGGTTAC) are specifically recognized by Pot1(1-389). Interestingly, certain single nucleotide substitutions that impacted Pot1pN binding exhibited no effect on binding affinity by Pot1(1-389). However, these substitutions reduced binding affinity when simultaneously substituted in each hexameric repeat. The non-additive nature of these substitutions suggests that certain nucleotides are coupled through the ability of the flexible ssDNA oligonucleotide to adopt alternate, thermodynamically equivalent conformations. The biochemical behavior of Pot1(1-389) is more similar to that of the full-length SpPot1 protein than to that of Pot1pN, making Pot1(1-389) a valuable domain for the future study of how full-length SpPot1 interacts with telomeric ssDNA.
机译：端粒1（Pot1）蛋白质的保护可以特异性识别端粒的单链3'端，这是持续维持细胞活力和增殖所必需的活性。粟酒裂殖酵母Pot1的端粒单链DNA（ssDNA）结合活性的当前模型基于20 kDa片段Pot1pN。最近的生化研究表明，SpPot1包含较大的ssDNA结合结构域，我们已经鉴定出了一个新的ssDNA结合结构域，其大小与人Pot1结构域相似。该结构域Pot1（1-389）与由两个保守的六聚体粟酒裂殖酵母端粒重复序列d（GGTTACGGTTAC）组成的寡核苷酸极其紧密地结合，其亲和力比与Pot1pN结合其同源ssDNA紧密约4000倍。 Pot1（1-389）/ ssDNA复合物的半衰期为53分钟，与全长SpPot1估计的半衰期一致，并且明显长于Pot1pN。单核苷酸取代显示，与Pot1pN相反，d（GGTTACGGTTAC）内的串联三核苷酸重复序列（GTT）被Pot1（1-389）特异性识别。有趣的是，某些影响Pot1pN结合的单核苷酸取代对Pot1（1-389）的结合亲和力没有影响。然而，当在每个六聚体重复中同时被取代时，这些取代降低了结合亲和力。这些取代的非累加性质表明，某些核苷酸通过柔性ssDNA寡核苷酸采用交替的，热力学上等效的构象的能力而偶联。 Pot1（1-389）的生物化学行为与全长SpPot1蛋白质的生物化学行为相比，与Pot1pN的生物化学行为更相似，这使得Pot1（1-389）成为将来研究全长SpPot1如何与之相互作用的有价值的领域。端粒ssDNA。

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