The Telomere Binding Protein Cdc13 and the Single-Stranded DNA Binding Protein RPA Protect Telomeric DNA from Resection by Exonucleases.
The Telomere Binding Protein Cdc13 and the Single-Stranded DNA Binding Protein RPA Protect Telomeric DNA from Resection by Exonucleases.
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DOI：
10.1016/j.jmb.2015.08.002
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发表时间：
2015-09-25
影响因子：
5.6
通讯作者：
Connolly BA
中科院分区：
文献类型：
--
作者：
Greetham M;Skordalakes E;Lydall D;Connolly BA
端粒存在于所有真核生物染色体的末端，通常由重复的富含TG的DNA组成，该DNA终止于单链的3‘TG延伸和一个富含5’CA的凹陷链。一种可以在体外观察核酸外切酶催化的端粒降解(切除)的生化分析方法已经被开发出来。该方法使用了一种寡聚脱氧核苷酸，它折叠成具有富含TG的双链区和3‘单链延伸的茎环，这是端粒的典型特征。端粒特异性Cst复合体的主要成分Cdc13强烈保护凹陷的链不受噬菌体→模型核酸外切酶5‘-λ和3’-核酸外切酶活性的影响。分离的CDC13的DNA结合域在屏蔽端粒方面的效果较差。保护是特异的，在缺乏特定富含甘油三酯的端粒序列的对照DNA中没有观察到。RPA是一种真核单链DNA结合蛋白，也能抑制端粒切除。然而，这种蛋白质是非特异性的，同样会阻碍非端粒对照的降解。端粒的自由端需要保护，以防止核酸酶催化的降解。CDC13是端粒特异性CST复合体的一种成分，它强烈地保护端粒。分离的CDC13的DNA结合域提供的保护作用弱于整个蛋白质。RPA是一种单链DNA结合蛋白，也是一种强大的端粒屏障。这项工作提供了第一个生化证据，证明CDC13和RPA都能够保护端粒免受核酸外切酶的影响。
The telomere is present at the ends of all eukaryotic chromosomes and usually consists of repetitive TG-rich DNA that terminates in a single-stranded 3′ TG extension and a 5′ CA-rich recessed strand. A biochemical assay that allows the in vitro observation of exonuclease-catalyzed degradation (resection) of telomeres has been developed. The approach uses an oligodeoxynucleotide that folds to a stem–loop with a TG-rich double-stranded region and a 3′ single-stranded extension, typical of telomeres. Cdc13, the major component of the telomere-specific CST complex, strongly protects the recessed strand from the 5′ → 3′ exonuclease activity of the model exonuclease from bacteriophage λ. The isolated DNA binding domain of Cdc13 is less effective at shielding telomeres. Protection is specific, not being observed in control DNA lacking the specific TG-rich telomere sequence. RPA, the eukaryotic single-stranded DNA binding protein, also inhibits telomere resection. However, this protein is non-specific, equally hindering the degradation of non-telomere controls. The free ends of telomeres need protection from nuclease-catalyzed degradation. Cdc13, one component of the telomere-specific CST complex, strongly protects telomeres. The isolated DNA binding domain of Cdc13 provides less protection than the entire protein. RPA, the single-stranded DNA binding protein, is also a powerful telomere shield. The work provides the first biochemical evidence that both Cdc13 and RPA are capable of protecting telomeres from exonuclease-catalyzed resection.