Recruitment of terminal protein to the ends of Streptomyces linear plasmids and chromosomes by a novel telomere-binding protein essential for linear DNA replication
Recruitment of terminal protein to the ends of Streptomyces linear plasmids and chromosomes by a novel telomere-binding protein essential for linear DNA replication
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DOI：
10.1101/gad.1060303
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发表时间：
2003-03-15
影响因子：
10.5
通讯作者：
Cohen, SN
中科院分区：
文献类型：
--
作者：
Bao, K;Cohen, SN
链霉菌线性质粒和染色体从中心起点的双向复制在复制中间体的端粒处产生未配对的T-前导链突出端。这些突出端的填充留下与成熟复制子的5' DNA末端共价连接的末端蛋白。我们在这里报告的一种新的80 kD的DNA结合蛋白（端粒相关蛋白，塔普）在这个过程中的重要作用。生物化学研究、酵母双杂交分析和免疫沉淀/免疫耗竭实验表明，Tap与3'突出端中的特定序列紧密结合，并且还与Tpg相互作用，将Tpg带到端粒末端。使用DNA微阵列分析染色体的自来水突变细菌，我们表明，自来水消融的幸存者进行端粒缺失，染色体环化，和扩增的亚端粒DNA。基于微阵列的染色体定位在单ORF分辨率揭示了独立缺失的共同终点，确定了扩增的染色体ORF邻近这些终点，并量化了这些ORF的拷贝数。序列分析证实了染色体环化，并揭示了外源DNA插入之间加入染色体末端。我们的结果表明，Tap是链霉菌线性DNA复制所必需的，并表明它的功能是招募和定位Tpg在复制中间体的端粒。他们还确定了伴随染色体环化的端粒缺失和亚端粒DNA扩增的热点。
Bidirectional replication of Streptomyces linear plasmids and chromosomes from a central origin produces unpaired T-leading-strand overhangs at the telomeres of replication intermediates. Filling in of these overhangs leaves a terminal protein attached covalently to the 5' DNA ends of mature replicons. We report here the essential role of a novel 80-kD DNA-binding protein (telomere-associated protein, Tap) in this process. Biochemical studies, yeast two-hybrid analysis, and immunoprecipitation/immunodepletion experiments indicate that Tap binds tightly to specific sequences in 3' overhangs and also interacts with Tpg, bringing Tpg to telomere termini. Using DNA microarrays to analyze the chromosomes of tap mutant bacteria, we demonstrate that survivors of Tap ablation undergo telomere deletion, chromosome circularization, and amplification of subtelomeric DNA. Microarray-based chromosome mapping at single-ORF resolution revealed common endpoints for independent deletions, identified amplified chromosomal ORFs adjacent to these endpoints, and quantified the copy number of these ORFs. Sequence analysis confirmed chromosome circularization and revealed the insertion of adventitious DNA between joined chromosome ends. Out results show that Tap is required for linear DNA replication in Streptomyces and suggest that it functions to recruit and position Tpg at the telomeres of replication intermediates. They also identify hotspots for the telomeric deletions and subtelomeric DNA amplifications that accompany chromosome circularization.