Identification of DNA-binding proteins that recognize a conserved type I repeat sequence in the replication origin region of Tetrahymena rDNA.
Identification of DNA-binding proteins that recognize a conserved type I repeat sequence in the replication origin region of Tetrahymena rDNA.
复制标题
鉴定识别四膜虫 rDNA 复制起点区域中保守的 I 型重复序列的 DNA 结合蛋白。
DOI：
10.1093/nar/22.21.4432
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发表时间：
1994
影响因子：
14.9
通讯作者：
Dobbs,DL
中科院分区：
文献类型：
--
作者：
Umthun,AR;Hou,Z;Sibenaller,ZA;Shaiu,WL;Dobbs,DL
在嗜热四膜虫（Tetrahymena thermophila）大核rRNA基因（rDNA）的5'端非转录间隔区（5' non-transcribed spacer region）定位了DNA复制起点。位于起始区域的33 nt保守的富含AT的I型重复序列中的突变导致体内扩增的rDNA的复制和/或维持的缺陷。Fe（II）EDTA切割足迹的限制性片段含有I型重复序列显示，大多数保守的核苷酸被保护的四膜虫细胞提取物中的蛋白质。在电泳迁移率变动分析中使用合成寡核苷酸鉴定并表征了结合I型重复序列的两类蛋白质。其中之一，ds-TIBF，优先结合双链体DNA和I型重复序列仅表现出中度特异性。相比之下，单链DNA结合蛋白，ssATIBF，特异性识别的A-丰富的链的I型重复序列。保守序列的5′或3′边界的缺失显著降低ssA-TIBF的结合。ssATIBF的结合特性，加上遗传证据表明，I型序列的功能作为C/S-作用rDNA复制控制元件在体内，建议ssA-TIBF在rDNA复制在四膜虫的可能作用。
An origin of DNA replication has been mapped within the 5' non-transcribed spacer region of the amplified macronuclear rRNA genes (rDNA) of Tetrahymena thermophila. Mutations in 33 nt conserved AT-rich Type I repeat sequences located in the origin region cause defects in the replication and/or maintenance of amplified rDNA in vivo. Fe(ll)EDTA cleavage footprinting of restriction fragments containing the Type I repeat showed that most of the conserved nucleotides were protected by proteins in extracts of Tetrahymena cells. Two classes of proteins that bound the Type I repeat were identified and characterized using synthetic oligonucleotides in electrophoretic mobility shift assays. One of these, ds-TIBF, bound preferentially to duplex DNA and exhibited only moderate specificity for Type I repeat sequences. In contrast, a single-stranded DNA-binding protein, ssATIBF, specifically recognized the A-rich strand of the Type I repeat sequence. Deletion of the 5′ or 3′ borders of the conserved sequence significantly reduced binding of ssA-TIBF. The binding properties of ssATIBF, coupled with genetic evidence that Type I sequences function as c/s-acting rDNA replication control elements in vivo, suggest a possible role for ssA-TIBF in rDNA replication in Tetrahymena.