Raghuraman M K, Dunn C J, Hicke B J, Cech T R
Department of Molecular, Cellular and Developmental Biology, Howard Hughes Medical Institute, Boulder, CO 80309-0215.
Nucleic Acids Res. 1989 Jun 12;17(11):4235-53. doi: 10.1093/nar/17.11.4235.
The telomere binding protein from macronuclei of Oxytricha nova binds macronuclear DNA in vitro, protecting the 3'-terminal single-stranded (T4G4)2 tail from chemical and enzymatic probes. We have used synthetic oligodeoxynucleotides to study the binding properties of the telomere protein. It binds at the 3' end of single-stranded oligonucleotides that have the sequence (T4G4)n, where n greater than or equal to 2, reconstituting the methylation protection seen with macronuclear DNA. Three oligonucleotide.protein complexes are resolved in nondenaturing gels, all specific for this sequence. Single-stranded oligonucleotides that have one or more repeats of the sequence C4A4 are also recognized, forming a single complex. The dissociation constant for (T4G4)4 is about 19 nM, and for macronuclear DNA is at least 20-fold lower. The basis for this difference is not fully understood, but it is not simply due to the absence of a (C4A4)2.5.(G4T4)2.5 region on the oligonucleotide. Transversions of T's to A's or of G's to C's in the 3' tail portion prevent binding. Changing T's to dU's does not prevent binding, indicating that the hydrophobic 5-methyl group is not required for binding as had been suggested from the salt-stability of the complex. The properties of the DNA-protein complex suggest a revised model for telomere synthesis in Oxytricha.
来自新大核草履虫大核的端粒结合蛋白在体外与大核DNA结合，保护3'端单链（T4G4）2尾巴免受化学和酶促探针的影响。我们使用合成寡脱氧核苷酸来研究端粒蛋白的结合特性。它结合在具有序列（T4G4）n（其中n大于或等于2）的单链寡核苷酸的3'端，重建了大核DNA所见的甲基化保护。在非变性凝胶中解析出三种寡核苷酸 - 蛋白质复合物，均对该序列具有特异性。具有一个或多个C4A4序列重复的单链寡核苷酸也被识别，形成单一复合物。（T4G4）4的解离常数约为19 nM，而大核DNA的解离常数至少低20倍。这种差异的基础尚未完全理解，但这不仅仅是由于寡核苷酸上不存在（C4A4）2.5.（G4T4）2.5区域。3'尾部部分中T向A或G向C的颠换会阻止结合。将T变为dU不会阻止结合，这表明如从复合物的盐稳定性所暗示的那样，结合不需要疏水的5 - 甲基基团。DNA - 蛋白质复合物的特性为新大核草履虫中端粒合成提出了一个修订模型。
