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回文序列是染色体的一个有效来源 断裂和重排，在疾病的发病机制中起重要作用。在人类身上，对于 例如，在非复发性和复发性的染色体断裂点发现了回文。 可能导致伊曼纽尔综合症的易位。此外，回文介导的染色体 重排导致几种类型的εγδβ地中海贫血、X连锁先天性多毛症综合征和 遗传性肾细胞癌。最后，回文与促进基因的放大有关 结肠癌、乳腺癌、髓母细胞瘤和淋巴瘤的肿瘤发生。尽管存在严重的影响， 关于基因组维护和疾病的回文，这些重复如何导致染色体断裂和 真核细胞中的重排在很大程度上是未知的。这项研究的总体目标是阐明 酿酒酵母回文序列中染色体断裂的机制。这个 该提议的中心假设是，回文序列中的染色体脆弱性是由多个 核酸酶在细胞周期的不同阶段，并取决于形成的二级结构的类型。我们的 初步数据表明，二级结构可以通过三条不同的途径启动双核糖核酸。 链断裂(DSB)形成并促进染色体不稳定。其中一条途径涉及 Mre11/Rad50/Xrs2(MRX)复合体和Sae2，依赖于结构特异性的MUS81/MMS4核酸酶， 一种涉及一种未知的核酸酶。在目标1中，我们将确定DSB形成所需的因素 MRX复合体和Sae2.我们发现，完全回文而不是准回文是DSB的位置 由MRX/Sae2形成，并将检验核酸酶攻击发夹结构的假设 S时期的完美回文。目标2将确定回文序列的参数，这些参数决定了 MUS81/MMS4核酸酶攻击二级结构的特异性。我们发现那件破损是在一家 由活跃转录的基因组成的完美回文部分依赖于MUS81/MMS4核酸酶。 这种核酸酶不会在另外两个非转录回文中中断。我们将确定 在MUS81/MMS4攻击中的转录，并将定义这种靶向所需的回文参数。目标 3将确定负责MRX/SAE2-和MUS81/MMS4-非依赖性回文断裂的蛋白质(S)。 存在由Lsm2-8复合体控制的回文脆弱性的另一种途径的假说 并对CDC28激酶进行测试。我们假设这条途径涉及一种未知的十字形分解酶 在细胞周期的G2阶段运行。这项拟议的研究具有创新性，因为它利用了 一套独特的敏感工具，将允许识别所有导致回文脆弱性的核酸酶 并将确定它们的切割特异性。这项建议意义重大，因为它将阐明- 明确了导致人类疾病的染色体异常的机制。