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有效来源 断裂和重排，并在人类疾病的发病机制中发挥重要作用。 例如，在非复发和复发的染色体断点处发现了回文 易位可导致伊曼纽尔综合征。此外，回文介导的染色体。 重排会导致多种类型的 εγδβ 地中海贫血、X 连锁先天性多毛症综合征和 最后，回文与促进基因的扩增有关。 尽管对结肠癌、乳腺癌、髓母细胞瘤和淋巴瘤的肿瘤发生有至关重要的影响。 回文对基因组维护和疾病的影响，这些重复如何导致染色体断裂以及 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重排。 酿酒酵母回文序列染色体断裂的机制。 该提案的中心假设是回文序列的染色体脆弱性是由多种原因引起的 细胞周期的不同阶段的核酸酶取决于所形成的二级结构的类型。 初步数据表明，二级结构可以通过三种不同的途径启动双 链断裂（DSB）形成并促进染色体不稳定的一种途径涉及。 Mre11/Rad50/Xrs2 (MRX) 复合体和 Sae2，一种取决于结构特异性 Mus81/Mms4 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 发现 DSB 的位点是完美回文，而不是准回文。 由 MRX/Sae2 形成，并将检验核酸酶攻击发生在由 MRX/Sae2 形成的发夹结构上的假设 S 阶段的完美回文将识别确定回文序列的参数。 Mus81/Mms4 核酸酶的二级结构攻击的特异性我们发现在 a 处发生断裂。 由活跃转录基因组成的完美回文部分依赖于 Mus81/Mms4 核酸酶。 该核酸酶不会在其他两个非转录回文结构上产生断裂，我们将确定其作用。 Mus81/Mms4 攻击中的转录，并将定义此类目标所需的回文参数。 图 3 将鉴定负责 MRX/Sae2 和 Mus81/Mms4 独立的回文断裂的蛋白质。 假设存在另一种由 Lsm2-8 复合体控制的回文脆弱性途径 我们将测试 Cdc28 激酶，因为该途径涉及未知的十字形解离酶。 所提出的研究是创新的，因为它利用了细胞周期的 G2 阶段。 一套独特的敏感工具，可以识别所有导致回文脆弱性的核酸酶 并将确定它们的裂解特异性。这个提议很重要，因为它将阐明较差的- 确定了产生人类疾病的染色体畸变的机制。