Telomeric protein-DNA point contacts identified by photo-cross-linking using 5-bromodeoxyuridine.
Telomeric protein-DNA point contacts identified by photo-cross-linking using 5-bromodeoxyuridine.
复制标题
使用 5-溴脱氧尿苷通过光交联鉴定端粒蛋白-DNA 点接触。
DOI：
10.1021/bi00177a030
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发表时间：
1994
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：
Hicke,BJ;Willis,MC;Koch,TH;Cech,TR
1993年12月30日收到的修订版Mandalpt ®摘要：Oxytricha端粒蛋白特异性识别单链端粒DNA，形成一种极其耐盐和动力学稳定的核蛋白复合物。关于这种异二聚体蛋白如何与DNA结合的信息的缺乏促使了这项光交联研究。在UV照射用终止于5 '-Ti6 T [5 T 14 T13 G12 GHG 10 G9 T8 T7 T6 T5 G4 G3 G2 G1 - 3'的合成寡核苷酸重构的尖孢属端粒时，形成多个蛋白质-DNA光交联。用5-溴脱氧尿苷（BrdU）对某些核苷酸进行位点特异性取代大大增加了光交联产率，每个取代有利于特定的蛋白质-DNA交联。例如，BrdU取代T7导致结合DNA的25%交联，比未取代的DNA增加10倍。端粒蛋白质的两个亚基都与DNA交联，因此靠近DNA。使用BrdU取代建立了该核蛋白复合物内涉及α亚基的三个点接触：Tyr 239、Tyr 142和His 292分别与G3、T15和T7交联。一个光交联，Tyr 239-G3，发生在a亚基的短酸性延伸中，与接近聚阴离子DNA的氨基酸的预期相反。剩余的两个交联是与一级多肽序列的疏水区域中的氨基酸的交联，这与疏水相互作用解释该蛋白质-DNA复合物的耐盐性（> 2 M NaCl）的假设一致。这两种光交联反应表明端粒蛋白可能通过将芳香族残基插入核苷酸晶格中与端粒单链DNA结合。端粒是真核生物染色体的物理末端，由典型的重复DNA和相关蛋白组成。端粒通过形成保护帽赋予染色体稳定性，所述保护帽阻止外切核酸溶解活性并防止在由染色体断裂产生的游离末端处发生的端对端融合。然而，端粒不是惰性的，而是必须动态地与反式作用因子相互作用以允许端粒的额外活动：染色体末端的完全复制和可能参与核结构。端粒的这些特征已被审查（Zakian，1989; Blackburn，1990，1991）。来自各种真核生物的端粒DNA序列都是相似的，由短的富G序列及其互补的富C链的串联重复序列组成（Blackburn，1991）。例如，脊椎动物端粒DNA由T2 AG 3的重复序列组成（Moyzis等人，（1988）单细胞
Revised Manuscript Received December 30, 1993® abstract: The Oxytricha telomere protein specifically recognizes single-stranded telomeric DNA, forming an extremely salt resistant and kinetically stable nucleoprotein complex. The absence of information on how this heterodimericprotein binds to DNA prompted this photo-cross-linking study. Multiple protein-DNA photo-cross-links are formed upon UV irradiation of Oxytricha telomeres reconstituted with a synthetic oligonucleotide terminating in 5'-Ti6T [5T 14T13G12GHG10G9T8T7T6T5G4G3G2G1-3'. Site-specific substitution of certain nucleotides with 5-bromodeoxyuridine (BrdU) greatly increased the photo-cross-linking yield, each substitution favoring a specific protein-DNA cross-link. For example, substitution of BrdU for T7 resulted in 25% cross-linking of the bound DNA, a 10-fold increase over the unsubstituted DNA. Both subunits of the telomere protein cross-link to, and are therefore near, the DNA. Three point contacts within this nucleoprotein complex, involving the a subunit, were established using BrdU substitution: Tyr239, Tyrl42, and His292 cross-link to G3, T15, and T7, respectively. One photo-cross-link, Tyr239-G3, occurs amid a short acidic stretch of the a subunit, counter to expectations for amino acids that approach the polyanionic DNA. The two remaining cross-links are to amino acids in hydrophobic regions of the primary polypeptide sequence, consistent with the hypothesis that hydrophobic interactions account for the salt resistance (> 2 M NaCl) of this protein-DNA complex. These two photo-cross-links suggest that the telomere protein may bind telomeric single-stranded DNA by intercalation of aromatic residues into a nucleotide lattice.Telomeres, the physical ends of eukaryotic chromosomes, consist of a canonical repetitive DNA and associated protein (s). Telomeres confer stability to chromosomes by forming a protective cap that deters exonucleolytic activities and prevents the end-to-end fusion that occurs at free ends generated by chromosome breakage. However, the telomere is not inert, but must dynamically interact with trans-acting factors to permit additional activities of telomeres: complete replication of the chromosomal terminus and possible participation in nuclear architecture. These features of telomeres have been reviewed (Zakian, 1989; Blackburn, 1990, 1991). Telomeric DNA sequences from a variety of eukaryotes are all similar, consisting of tandem repeats of a short G-rich sequence and its complementary C-rich strand (Blackburn, 1991). For example, vertebratetelomeric DNA consists of repeats of T2AG3 (Moyzis et al., 1988), and the unicellular