A new model for Schizosaccharomyces pombe telomere recognition:: The telomeric single-stranded DNA-binding activity of Pot11-389
A new model for Schizosaccharomyces pombe telomere recognition:: The telomeric single-stranded DNA-binding activity of Pot11-389
复制标题
DOI：
10.1016/j.jmb.2006.06.002
复制
发表时间：
2006-08-04
影响因子：
5.6
通讯作者：
Wuttke, Deborah S.
中科院分区：
文献类型：
--
作者：
Croy, Johnny E.;Podell, Elaine R.;Wuttke, Deborah S.
端粒1（Pot 1）蛋白的保护特异性地识别端粒的单链3'端，这是持续细胞活力和增殖所必需的活性。粟酒裂殖酵母Pot 1端粒单链DNA（ssDNA）结合活性的当前模型是基于20 kDa片段Pot 1 pN。最近的生化研究表明，SpPot 1包含一个更大的ssDNA结合域，我们已经确定了一个新的ssDNA结合域的大小类似于人类Pot 1域。该结构域Pot 1（1-389）与由两个保守的六聚体S.粟酒裂殖酵母端粒重复序列，d（GGTTACGGTTAC），其亲和力比Pot 1 pN结合其同源ssDNA的亲和力强4000倍。Pot 1（1-389）/ssDNA复合物的半衰期为53 min，与全长SpPot 1的估计值一致，明显长于Pot 1 pN。单核苷酸取代表明，与Pot 1 pN相反，d（GGTTACGGTTAC）内的串联三核苷酸重复序列（GTT）被Pot 1（1-389）特异性识别。有趣的是，某些影响Pot 1 pN结合的单核苷酸取代对pot 11 -389的结合亲和力没有表现出任何影响。然而，当在每个六聚体重复序列中同时取代时，这些取代降低了结合亲和力。这些取代的非加性性质表明，某些核苷酸通过柔性ssDNA寡核苷酸采用交替的、化学上等同的构象的能力偶联。potll-389的生物化学行为与全长SpPot 1蛋白比Pot 1 pN更相似，使得potll-389成为未来研究全长SpPot 1如何与端粒ssDNA相互作用的有价值的结构域。(c)2006爱思唯尔有限公司保留所有权利。
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 similar to 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 potll-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 potll-389 is more similar to that of the full-length SpPot1 protein than to that of Pot1pN, making potll-389 a valuable domain for the future study of how full-length SpPot1 interacts with telomeric ssDNA. (c) 2006 Elsevier Ltd. All rights reserved.