Hotspots of de novo telomere addition as mediators of genomic instability in yeast

项目摘要 端粒是高度重复的、蛋白质结合的、富含G的序列，覆盖了大多数真核细胞染色体的末端。 嵌合体。作为核糖核蛋白复合体端粒酶的底物，端粒提供了一种机制 抵消复制过程中发生的末端DNA的渐进性丢失。端粒也区分了 正常的染色体末端来自需要修复的断端。不适当地招募修复蛋白来 正常的染色体末端允许形成双着丝粒，从而对细胞活力产生灾难性的后果 染色体是末端融合事件的结果。 端粒酶有能力在双链之后的内部位置产生新的功能端粒 中断(DSB)。这种从头添加端粒可通过产生末端干扰正常修复 删除。事实上，染色体截断，其中一些是由于端粒的从头增加而产生的，是一种 人类遗传病的常见来源。考虑到这些后果，细胞具有 进化的机制，以抑制端粒酶在DSB的作用。我们实验室最近发表的研究成果 已经鉴定了酵母基因组中的两个序列，它们支持更高频率的从头端粒- 仅比邻近区域增加[称为SIRTA(内修复相关端粒增加的部位- Tion)]。我们假设SiRTA对正常情况下抑制端粒酶活性的机制不起作用。 DSB由于特定的顺式和反式作用因子和/或因为端粒酶作用不再被抑制 当断裂持续未修复时。在这里，我们描述了导致高频的因素 并检测端粒酶在这些基因组热点上的作用 不稳定。我们的实验在解决内源性位点的端粒添加调节方面是独一无二的 可能位于起始DSB的数百或数千个核苷酸中。 尽管端粒从头添加的有害后果是显而易见的，但基因组的分布 在任何生物体中，都没有确定具有异常高的调节这类事件的潜力的位置。在她身上 20世纪40年代的开创性工作，芭芭拉·麦克林托克第一个提出端粒的增加可以 用来限制染色体断裂，从而防止染色体在面对原本无法修复的堤坝时丢失- 年龄。目前尚不清楚端粒添加的基因组热点是否是选择其他 功能，或者可能已经进化或被保留为DNA损伤耐受的机制。系统化的 酵母中siRTA的鉴定将提供工具来回答这个问题，同时强调以下特征 通过端粒从头加成导致基因组不稳定。

Project Summary Telomeres are highly repetitive, protein-bound, G-rich sequences that cap the ends of most eukaryotic chro- mosomes. As substrates for the ribonucleoprotein complex telomerase, telomeres provide a mechanism to counteract the progressive loss of terminal DNA that occurs during replication. Telomeres also distinguish normal chromosome ends from broken ends in need of repair. Inappropriate recruitment of repair proteins to normal chromosome ends has disastrous consequences for cell viability by allowing formation of dicentric chromosomes as a result of end fusion events. Telomerase has the ability to create a new, functional telomere at an internal site following a double-strand break (DSB). Such de novo telomere addition can interfere with normal repair through the creation of terminal deletions. Indeed, chromosome truncations, some of which arise through de novo telomere addition, are a common source of human genetic disease. Given these consequences, it is not surprising that cells have evolved mechanisms to inhibit the action of telomerase at DSBs. Work recently published from our laboratory has characterized two sequences in the yeast genome that support much higher frequencies of de novo telo- mere addition than neighboring regions [termed SIRTAs (Sites of Internal Repair-associated Telomere Addi- tion)]. We hypothesize that SiRTAs are refractory to mechanisms that normally inhibit telomerase action at a DSB due either to specific cis- and trans-acting factors and/or because telomerase action is no longer inhibited when a break has persisted without repair. Here we characterize factors that contribute to the high frequency of telomere addition at SiRTAs and examine the regulation of telomerase action at these hotspots of genomic instability. Our experiments are unique in addressing the regulation of telomere addition at endogenous sites that may be located hundreds or thousands of nucleotides from the initiating DSB. Although the detrimental consequences of de novo telomere addition are clear, the distribution of genomic sites with abnormally high potential to mediate such events has not been determined in any organism. In her groundbreaking work in the 1940s, Barbara McClintock was the first to suggest that telomere addition could serve to cap a chromosome break, thus preventing chromosome loss in the face of otherwise irreparable dam- age. It is currently unclear whether genomic hotspots of telomere addition are a byproduct of selection for other functions, or may have evolved or been retained as a mechanism of DNA damage tolerance. The systematic identification of SiRTAs in yeast will provide tools to answer this question while highlighting the features that lead to genomic instability through de novo telomere addition.