CHARACTERIZATION OF A G-QUARTET FORMATION REACTION PROMOTED BY THE BETA-SUBUNIT OF THE OXYTRICHA TELOMERE-BINDING PROTEIN
CHARACTERIZATION OF A G-QUARTET FORMATION REACTION PROMOTED BY THE BETA-SUBUNIT OF THE OXYTRICHA TELOMERE-BINDING PROTEIN
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DOI：
10.1021/bi00094a022
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发表时间：
1993-11-02
期刊：
影响因子：
2.9
通讯作者：
CECH, TR
中科院分区：
文献类型：
--
作者：
FANG, GW;CECH, TR
端粒是线性染色体的末端，通常由简单的富含鸟嘌呤序列的串联重复序列组成。端粒DNA能够形成分子间G-四联体结构。尖孢属端粒结合蛋白的β亚基作为分子伴侣，促进由G-四联体稳定的端粒DNA的二聚体和特定的高阶复合物的形成;这些反应在体外生理条件下发生。在本文中，我们表明，在饱和蛋白质浓度（大于或等于200 nM），β介导的G-四联体形成是一个一级反应相对于DNA浓度，与k几乎等于1小时-1在37 ℃。相反，蛋白质非依赖性反应是二级反应。在端粒DNA浓度为20 nM时，β-亚基使G-四联体形成速率提高10（5）-10（6）倍。β介导的高阶复合物被鉴定为端粒DNA的平行四链四聚体（G4-DNA）。聚-L-赖氨酸也促进四聚体的形成，但不促进二聚体的形成。这些DNA结构进行了研究，通过不可逆的热熔融实验和退火不同的互补链探测。通过甲基化干扰实验分析了对结构形成重要的鸟嘌呤残基。在这些数据的基础上，提出了β-介导的结构模型，并讨论了β-介导的反应的可能机制。此外，我们发现β亚基促进两条互补链退火成双链体，就像许多其他碱性蛋白质一样。然而，并不是所有具有退火促进活性的蛋白质都在G-四重体结构的形成中具有活性。端粒蛋白在促进端粒DNA结构形成中的活性可能使染色体-染色体缔合或体内端粒酶活性的调节成为可能。
Telomeres, the ends of linear chromosomes, typically consist of tandem repeats of a simple guanine-rich sequence. Telomeric DNA is able to form intermolecular G-quartet structures. The beta-subunit of the Oxytricha telomere-binding protein acts as a molecular chaperone to promote the formation of dimers and specific higher order complexes of telomeric DNA stabilized by G-quartets; these reactions occur under physiological conditions in vitro. In the present article, we show that, at saturating protein concentrations (greater-than-or-equal-to 200 nM), beta-mediated G-quartet formation is a first-order reaction with respect to DNA concentration, with k almost-equal-to 1 h-1 at 37-degrees-C. In contrast, the protein-independent reaction is a second-order reaction. The beta-subunit enhances the rate of G-quartet formation by 10(5)-10(6)-fold at a telomeric DNA concentration of 20 nM. The beta-mediated higher order complexes are identified as parallel four-stranded tetramers of telomeric DNA (G4-DNA). Poly-L-lysine also promotes formation of the tetramers, but not dimers. These DNA structures were studied by irreversible thermal melting experiments and probed by annealing to different complementary strands. Guanine residues important for structure formation were analyzed by methylation interference experiments. On the basis of these data, models for the beta-mediated structures are proposed, and possible mechanisms for the beta-mediated reaction are discussed. In addition, we found that the beta-subunit promotes the annealing of two complementary strands into a duplex, as do many other basic proteins. However, not all proteins with annealing-promoting activity are active in the formation of G-quartet structures. The activity of the telomere protein in promoting the formation of telomeric DNA structures may enable chromosome-chromosome association or the regulation of telomerase activity in vivo.