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

从头端粒添加的热点作为酵母基因组不稳定性的介质

• 批准号: 9290115
• 负责人: Katherine Louise Friedman
• 金额: $ 32.94万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9290115
• 关键词: Address; Affect; Attenuated; Binding; Binding Proteins; Binding Sites; Cell Survival; Cell division; Cells; Chromosomal Breaks; Chromosomal Stability; Chromosome Arm; Chromosome Breakage; Chromosome abnormality; Chromosomes; Cis-Acting Sequence; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; DNA; DNA Damage; DNA Repair; DNA-Binding Proteins; Defect; Development; Dicentric chromosome; Dimerization; Dinucleoside Phosphates; Disease; Distal; Distant; Double Strand Break Repair; Enzymes; Event; Excision; Failure; Frequencies; Functional disorder; Genes; Genetic Screening; Genome Stability; Genomic DNA; Genomic Instability; Genomics; Goals; Growth; Hereditary Disease; Human Genetics; Impairment; Individual; Laboratories; Lead; Location; Malignant Neoplasms; Mediating; Mediator of activation protein; Methods; Modeling; Monitor; Mutation; N-terminal; Names; Nucleotides; Organism; Pathway interactions; Population; Proteins; Publications; Publishing; Recruitment Activity; Refractory; Regulation; Reporting; Ribonucleoproteins; Role; Seeds; Site; Source; Stem cells; Survivors; TERT gene; Telomerase; Testing; Time; Trans-Activators; Work; Yeasts; base; cancer cell; chromosome loss; deep sequencing; dimer; endonuclease; experimental study; insight; interest; prevent; protein structure; repaired; response; telomere; tool; yeast genome

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.

项目摘要 端粒是高度重复的、蛋白质结合的、富含G的序列，其覆盖大多数真核生物染色体的末端。 摩梭人作为核糖核蛋白复合物端粒酶的底物，端粒提供了一种机制， 抵消在复制过程中发生的末端DNA的逐渐丢失。端粒也能区分 正常的染色体末端从需要修复的断裂末端。修复蛋白的不适当募集， 正常的染色体末端由于允许形成双着丝粒， 染色体作为结束融合事件的结果。 端粒酶具有在双链断裂后的内部位点产生新的功能性端粒的能力。 中断（DSB）。这样的从头端粒添加可以通过末端端粒的产生来干扰正常的修复。 删除。事实上，染色体截短，其中一些是通过从头端粒增加而产生的，是一种遗传学上的疾病。 人类遗传疾病的共同来源。考虑到这些后果，细胞具有 进化出抑制端粒酶在DSB中作用的机制。我们实验室最近发表的研究成果 已经表征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， 与邻近区域相比，仅仅增加了[称为SIRTA（内部修复相关端粒附加位点）， ）]。我们假设SiRTAs对正常情况下抑制端粒酶活性的机制是难治的， 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受到抑制 当破裂持续而不修复时。在这里，我们的特点因素，有助于高频率 研究端粒在SiRTAs上的添加，并检查端粒酶在这些基因组热点的调节作用。 不稳定我们的实验是独特的解决调节端粒添加在内源性网站 其可以位于距起始DSB数百或数千个核苷酸处。 虽然从头添加端粒的有害后果是清楚的，但基因组端粒的分布是不确定的。 在任何生物体中都没有确定具有异常高的介导这种事件的可能性的位点。在她 在20世纪40年代的开创性工作中，芭芭拉·麦克林托克是第一个提出端粒增加可以 用于覆盖染色体断裂，从而防止染色体丢失，否则无法修复的大坝， 年龄目前尚不清楚端粒增加的基因组热点是否是其他选择的副产品。 功能，或者可能已经进化或保留为DNA损伤耐受机制。系统 酵母中SiRTA的鉴定将提供回答这个问题的工具，同时突出显示 通过从头添加端粒导致基因组不稳定性。