Identification of distinct pathways for DSB formation at palindromic repeats

回文重复 DSB 形成的不同途径的鉴定

• 批准号: 9922336
• 负责人: KIRILL S LOBACHEV
• 金额: $ 30.27万
• 依托单位: GEORGIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9922336
• 关键词: Adopted; Animal Model; Cell Cycle Stage; Cells; Chromosomal Instability; Chromosomal Rearrangement; Chromosome Breakage; Chromosome Fragility; Chromosome abnormality; Colon Carcinoma; Complex; DNA Repair Pathway; Data; Disease; Double Strand Break Repair; Drug Design; Embryo; Etiology; Eukaryotic Cell; Fission Yeast; Gene Amplification; Generations; Genes; Genetic; Genetic Recombination; Genetic Transcription; Genome; Goals; Health; Hereditary Renal Cell Carcinoma; Holliday Junction Resolvases; Human; Human Genome; Hypertrichosis; Inherited; Life; Link; Lymphoma; Maintenance; Mediating; Mission; Molecular; Organism; Pathogenesis; Pathology; Pathway interactions; Phosphotransferases; Play; Predisposition; Proteins; Public Health; Recurrence; Research; Resolvase; Role; S Phase; Saccharomyces cerevisiae; Site; Source; Specificity; Structure; Syndrome; Testing; Thalassemia; Translocation Breakpoint; United States National Institutes of Health; Work; Yeasts; burden of illness; cancer cell; disability; endonuclease; human disease; improved; innovation; kidney cell; malignant breast neoplasm; medulloblastoma; mutant; novel; nuclease; outcome forecast; tool; tumorigenesis; tumorigenic

Palindromic sequences that adopt hairpin and cruciform structures are a potent source of chromosomal breakage and rearrangements, and play a significant role in the pathogenesis of diseases. In humans, for example, palindromes have been found at chromosomal breakpoints of non-recurrent and recurrent translocations that can cause Emanuel syndrome. In addition, palindrome-mediated chromosomal rearrangements cause several types of εγδβ thalassemia, X-linked congenital hypertrichosis syndrome and hereditary renal cell carcinoma. Finally, palindromes are implicated in the amplification of genes that promote tumorigenesis in colon and breast cancer, medulloblastoma and lymphoma. Despite the critical impact of palindromes on genome maintenance and disease, how these repeats cause chromosome breakage and rearrangements in eukaryotic cells is largely unknown. The overall objective of this research is to elucidate the mechanisms of chromosomal breakage at palindromic sequences in yeast, Saccharomyces cerevisiae. The central hypothesis of the proposal is that chromosomal fragility at palindromic sequences is caused by multiple nucleases at different stages of the cell cycle and is dependent on the type of secondary structure formed. Our preliminary data indicate that there are three distinct pathways by which secondary structures can initiate double- strand break (DSB) formation and promote chromosomal instability. One pathway involves the Mre11/Rad50/Xrs2 (MRX) complex and Sae2, one depends on the structure-specific Mus81/Mms4 nuclease, and one involves an unknown nuclease. In Aim 1, we will determine factors required for DSB formation by the MRX complex and Sae2. We have found that perfect palindromes but not quasi-palindromes are sites for DSB formation by MRX/Sae2 and will test the hypothesis that nuclease attack occurs on hairpin structures formed by perfect palindromes during S-phase. Aim 2 will identify parameters of palindromic sequences that determine the specificity of secondary-structure attack by the Mus81/Mms4 nuclease. We have found that breakage at a perfect palindrome composed of actively transcribed genes is partially dependent on the Mus81/Mms4 nuclease. This nuclease does not make breaks at two other nontranscribed palindromes. We will determine the role of transcription in Mus81/Mms4 attack and will define parameters of palindromes required for such targeting. Aim 3 will identify the protein(s) responsible for MRX/Sae2- and Mus81/Mms4-independent breakage of palindromes. The hypothesis that there is another pathway for palindrome fragility that is controlled by the Lsm2-8 complex and the Cdc28 kinase will be tested. We hypothesize that this pathway involves an unknown cruciform resolvase that operates during the G2 stage of the cell cycle. The proposed research is innovative because it utilizes a unique set of sensitive tools that will allow the identification of all nucleases contributing to palindrome fragility and will determine their cleavage specificity. This proposal is significant because it will elucidate the poorly- defined mechanisms that generate chromosomal aberrations that underlie human diseases.

采用发夹和十字形结构的后验序序列是染色体的潜在来源 断裂和重排，并在疾病的发病机理中起重要作用。在人类中 例如，在非染色和复发的染色体断裂点上发现了allindromes 可能导致伊曼纽尔综合征的易位。此外，腔介导的染色体 重排会引起几种类型的εγδβthalassyamia，X连锁的先天性肥大综合征和 遗传性肾细胞癌。最后，在促进的基因的扩增中暗示了alindromes 结肠癌和乳腺癌，髓母细胞瘤和淋巴瘤中的肿瘤发生。尽管有关键的影响 关于基因组维持和疾病的全文，这些重复如何导致染色体断裂和 真核细胞中的重排基本上未知。这项研究的总体目的是阐明 在酵母中葡萄球菌酿酒酵母中的全粒序列上的染色体破裂的机理。 该提案的中心假设是，plindromic序列处的染色体脆弱性是由多个引起的 核酸酶在细胞周期的不同阶段，取决于形成的二级结构的类型。我们的 初步数据表明，二级结构可以启动双重途径有三种不同的途径 链断裂（DSB）形成并促进染色体不稳定性。一条途径涉及 MRE11/RAD50/XRS2（MRX）复合物和SAE2，取决于结构特异性MUS81/MMS4核酸酶， 一个涉及未知的核酸酶。在AIM 1中，我们将确定DSB形成所需的因素 MRX复合物和SAE2。我们发现，完美的回文，而不是准palindromes是DSB的网站 由MRX/SAE2形成，并将检验以下假设：核酸酶攻击发生在由发夹结构上 S阶段期间的完美回文。 AIM 2将识别确定的后粒细胞序列的参数 MUS81/MMS4核酸酶的二级结构攻击的特异性。我们发现在 由主动转录基因组成的完美回文部分部分取决于MUS81/MMS4核酸酶。 这种核酸酶不会在另外两个非翻译的全文中休息。我们将确定 MUS81/MMS4攻击中的转录，并将定义此类靶向所需的alind骨参数。目的 3将确定负责MRX/SAE2-和MUS81/MMS4独立于alentlomes的蛋白质。 假设有另一个由LSM2-8复合物控制的腔杂志脆弱性的途径 并将测试Cdc28激酶。我们假设该途径涉及未知的十字形分辨率 在细胞周期的G2阶段运行。拟议的研究具有创新性，因为它利用了 独特的敏感工具集将允许识别所有造成引文脆弱性的核武器 并将确定其乳沟特异性。该提议很重要，因为它将阐明不良的 定义的机制产生了人类疾病构成的染色体畸变。