Recombination and Regulation

重组与调控

• 批准号: 8348991
• 负责人: Jeffrey Strathern
• 金额: $ 72.23万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8348991
• 关键词: Address; Adopted; Area; Bacteria; Base Sequence; Cells; Characteristics; Cloning; Collaborations; Computer Simulation; Computer software; DNA; DNA Sequence; DNA Sequence Rearrangement; DNA Structure; DNA analysis; DNA biosynthesis; Event; Gene Amplification; Generations; Genes; Genetic Recombination; Genome; Genome Stability; Genomic Instability; Goals; Head; Human; Human Genome; Laboratories; MCF7 cell; Mammary Neoplasms; Methods; Modeling; Normal Cell; Pathway interactions; Problem Solving; Production; Recombinants; Recovery; Regulation; Research; Sampling; Structure; System; Techniques; Testing; Tumor Cell Line; Work; Yeasts; base; cancer cell; expectation; mammalian genome; neoplastic cell; novel; novel diagnostics; novel strategies; novel therapeutics; research study; tool; tumor

The Genome Recombination/Regulation Section focuses on two topics related to recombination and genome stability: mechanisms that generate DNA palindromic gene amplifications and the origin of DNA synthesis errors associated with genetic recombination. Studies on genome instability. We are continuing our analysis of how DNA palindromes are generated. These head to head DNA sequences are highly unstable. Some tumor cells undergo gene amplification by unknown mechanisms that generate palindromes. The instability of these sequences contributes to additional genome rearrangements that occur in tumors. Because palindromes are unstable in bacteria, it is it nearly impossible to clone them. Similarly, the secondary structures that can be adopted by palindromic DNAs make them very difficult to sequence. We opened the field of research on the origin of DNA palindromes by making progress in three important areas related to the study of palindromes. First, we identified yeast strains that tolerate palindromes. Second, we developed a method that allows us to sequence palindromic DNAs. Third, we developed a recombination substrate that generates palindromes and identified a class of recombinants that is almost exclusively palindromes. We demonstrated that the palindromes are formed in our system by a novel kind of nonhomologous end joining (NHEJ) which is independent of some of the recombination functions that are required for most NHEJ events. We recently demonstrated that we can isolate palindromic sequences from mammalian genomes, opening the door to the analysis of palindromes found in normal and malignant cells. We are collaborating on the analysis of DNA palindromes and inverted repeats found in human tumors. This common mechanism of gene amplification in tumors was not accessible to physical characterization until the breakthroughs we made described above. We have developed new methods to isolate the novel junctions associated with DNA palindromes found in tumors. It is our expectation that the characterization of those junctions will help reveal details of the mechanism by which they are generated. Our similar approach to DNA palindromes in yeast was paradigm shifting in that it revealed a very different mechanism of formation quite unlike the generally accepted model. This year we applied high throughput sequencing to the fast annealing fraction of a human tumor cell line. This so called Cot0 DNA includes highly reiterated DNAs and inverted repeats. Our focus is on the inverted repeat fraction. Our analysis demonstrates that some regions of the genome are present as inverted repeats in the human breast tumor cell line MCF7 which are not inverted repeats in normal human cells. This analysis paves the way for us to isolate the novel junctions found in these inverted repeats to test the hypothesis that these structures result from a foldback priming mechanism. Our demonstration that foldback priming causes similar inverted repeats in yeast was the first new discovery of this pathway. Our goal is to investigate whether this mechanism represents an important pathway for gene amplification in tumor cells. Many of the new generation of techniques used to sequence DNAs involve PCR steps. Our research this year demonstrated that this approach systematically excludes the recovery of DNA palindromes because of the requirement for strand displacement when amplifying sequences that can form secondary structures. We generated a unique substrate with a 2 kb DNA palindrome and showed that current second generation sequencing approaches fail on that substrate. These results clearly show that the current in silico representation of the human genome was generated with techniques that could fail to accurately or completely determine the content and structure of the human genome. We are now developing new approaches to solving this problem that will allow the identification and sequencing of DNA palindromes in normal and malignant cells. We initiated a collaborative project with PacBio and the ATP Sequencing Facility to evaluate that approach to sequencing DNA palindromes. These experiments had two important results. First to demonstrate that the software used by PacBio could not readily recognize the novel junctions in palindromes. Second, when that problem was fixed, it is clear that PacBio can successfully sequence DNA palindromes. We are engaged with PacBio in extending these results to provide a general method of identifying and sequencing DNA palindromes in human tumor samples.

基因组重组/调节部分侧重于与重组和基因组稳定性有关的两个主题：产生DNA腔内粒细胞基因扩增的机制以及与基因重组相关的DNA合成误差的起源。 基因组不稳定性的研究。我们正在继续分析如何生成DNA palindromes。这些头到头DNA序列高度不稳定。一些肿瘤细胞通过产生allindromes的未知机制进行基因扩增。这些序列的不稳定性导致肿瘤中发生的其他基因组重排。由于细菌中的alend膜不稳定，因此几乎不可能克隆它们。同样，alindromic DNA可以采用的二级结构使它们难以顺序。我们通过在与本文综合体研究有关的三个重要领域取得进展，开辟了有关DNA alindromes起源的研究领域。首先，我们确定了耐触诊的酵母菌菌株。其次，我们开发了一种方法，使我们能够对后验元DNA进行测序。第三，我们开发了一种重组底物，该底物生成alindromes并确定了几乎完全是全文的重组者。我们证明了本文是通过一种新型的非同源末端连接（NHEJ）形成的，该末端与大多数NHEJ事件所需的某些重组函数无关。我们最近证明，我们可以从哺乳动物基因组中分离出腔素序列，从而打开了在正常和恶性细胞中发现的alind膜分析的大门。 我们正在协作对DNA palindromes的分析和人类肿瘤中发现的倒重复。直到我们在上面描述的突破之前，这种肿瘤中基因扩增的常见机制才能获得物理表征。我们开发了新的方法来隔离与肿瘤中发现的DNA palindromes相关的新连接。我们期望这些连接的表征将有助于揭示其生成的机制的细节。我们对酵母中DNA palindromes的类似方法是范式的转移，因为它揭示了一种非常不同的形成机理，这与普遍接受的模型完全不同。今年，我们将高通量测序应用于人类肿瘤细胞系的快速退火部分。所谓的COT0 DNA包括高度重复的DNA和倒重复序列。我们的重点是倒重复分数。我们的分析表明，基因组的某些区域存在于人类乳腺肿瘤细胞系MCF7中的反复重复序列，而正常人细胞中没有反转重复序列。该分析铺平了为我们隔离这些倒置重复序列中发现的新连接的方式，以检验这些结构是由折叠式启动机制引起的假设。我们证明折叠式启动会导致酵母中相似的倒重复序列，这是该途径的第一个新发现。我们的目标是研究这种机制是否代表了肿瘤细胞基因扩增的重要途径。用于对DNA进行测序的许多新一代技术都涉及PCR步骤。我们今年的研究表明，这种方法系统地排除了DNA palindromes的恢复，因为在放大可以形成二级结构的序列时需要链位移。我们生成了一个具有2 kb DNA腔室的唯一底物，并表明当前的第二代测序方法在该基材上失败了。这些结果清楚地表明，人类基因组的硅硅表示电流是通过可能无法准确或完全确定人类基因组的含量和结构的技术产生的。现在，我们正在开发解决此问题的新方法，该方法将允许正常和恶性细胞中DNA palindromes的鉴定和测序。我们与PACBIO和ATP测序设施启动了一个协作项目，以评估该测序DNA palindromes的方法。这些实验有两个重要的结果。首先证明PACBIO使用的软件无法轻易识别出回文中的新连接。其次，当解决该问题时，很明显PACBIO可以成功地对DNA palindromes进行序列。我们参与PACBIO扩展这些结果，以提供一种通用的方法，以鉴定和测序人类肿瘤样品中的DNA palindromes。