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DNA Misfolding and the Maintenance of Genome Stability: an Integrated Molecular, Cellular and Genomic Investigation of DNA Double-Strand Break Repair

DNA 错误折叠和基因组稳定性的维持：DNA 双链断裂修复的分子、细胞和基因组综合研究

基本信息

批准号：
MR/M019160/1
负责人：
David Leach
金额：
$ 219.76万
依托单位：
University of Edinburgh
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2015
资助国家：
英国
起止时间：
2015 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=MR%2FM019160%2F1
关键词：
DNA Misfolding Maintenance Genome Stability

项目摘要

DNA is essential for all living organisms as it encodes the information needed for growth, survival and inheritance. Chromosomes contain this genetic information in very long molecules of DNA (often more that several million molecular building blocks, known as base-pairs, in length). One consequence of this great length is fragility of the molecule. Therefore, cells need efficient systems to repair broken DNA. One single unrepaired DNA break in a chromosome leads to cell death. Not only does a break need to be repaired efficiently but it must also be repaired accurately. Inaccurate repair leads to genetic alterations that cause cancer or genetic disease. Furthermore, DNA breakage is used to kill cancer cells in treatments such as radiotherapy and chemotherapy. The break repair pathways also offer targets for the development of new antibiotics. Understanding how DNA molecules are broken and how they are repaired is fundamentally important to inform future clinical practice.Interestingly, not all DNA sequences are equally susceptible to breakage. Due to their inverted-repeat nature, palindromic DNA sequences can misfold in chromosomes and these misfolded structures have an elevated probability of breakage. DNA palindromes are known to be locations in chromosomes associated with human genetic disease in foetuses and cancer in adults. We know also that this association with disease is likely to be caused by DNA breakage. In bacteria (such as E. coli), we have discovered that a specific protein, known as SbcCD (also present in humans where it is known as Rad50/Mre11), is responsible for generating DNA breaks at palindromic sequences. However, these breaks are very efficiently and accurately repaired in a reaction called homologous recombination, using the genetic information on a second unbroken copy of the chromosome present in the same cells. We will use this system of DNA misfolding and cleavage (and another system that uses molecular scissors) to generate repairable DNA breaks at a specific chromosomal location and study their repair by homologous recombination. We will concentrate on the mechanism of repair of DNA breaks in living cells. This is the area where the least is known and where our research can have the greatest impact. Many years of genetic investigation have identified the genes involved in homologous recombination and the proteins that they encode have been studied in the test tube. However, the experimental systems to study this reaction in living cells and the methods to carry out this analysis have recently progressed to a stage where substantial progress can be made. Using microscopy, we will actually look at the repair reactions, as they are happening in live cells. We will also isolate and analyse the DNA molecules "caught in the act" of repairing breaks. Finally we will observe the indirect consequences of DNA break repair on the duplication of chromosomes and their distribution into the next generation of cells.Our sophisticated systems for making DNA breaks and analysing their repair, combined with the wealth of knowledge of E. coli as an experimental organism and its intrinsic advantages (such as small size and rapid growth rate) will be used to make more rapid progress than it is possible with more complex organisms. Not only will this work elucidate how DNA breaks are actually repaired in living E. coli (an important bacterial pathogen) but it will be a first step towards implementing similar experimental strategies in human cells. An example will help to illustrate this. We intend for the first time to isolate the DNA from a single DNA repair site on the chromosome and analyse this DNA by electron microscopy. This will not be trivial even for a chromosome of 4.6 million base pairs in length. However if we succeed, we will have pioneered the way towards a similar goal for the DNA in a human cell where a specific repair event will have to be isolated from over 4 billion base pairs of DNA.

DNA对所有生物来说都是必不可少的，因为它编码了生长、生存和遗传所需的信息。染色体在很长的DNA分子中包含这种遗传信息(通常在长度上超过几百万个分子构件，称为碱基对)。如此长的长度的一个后果是分子的脆弱性。因此，细胞需要有效的系统来修复断裂的DNA。染色体中一个未修复的DNA断裂就会导致细胞死亡。断裂不仅需要有效地修复，而且还必须准确修复。不准确的修复会导致基因改变，从而导致癌症或遗传病。此外，DNA断裂在放射治疗和化疗等治疗中被用来杀死癌细胞。断裂修复途径也为新抗生素的开发提供了靶点。了解DNA分子是如何被破坏的，以及它们是如何被修复的，对于未来的临床实践是至关重要的。有趣的是，并不是所有的DNA序列都同样容易被破坏。由于其反向重复的性质，回文DNA序列可能在染色体中错误折叠，并且这些错误折叠的结构具有更高的断裂概率。DNA回文是已知的与人类胎儿遗传病和成人癌症相关的染色体上的位置。我们还知道，这种与疾病的联系很可能是由DNA断裂引起的。在细菌(如大肠杆菌)中，我们发现一种名为SbcCD的特定蛋白质(在人类中也存在，它被称为Rad50/Mre11)，负责在回文序列上产生DNA断裂。然而，这些断裂在一种称为同源重组的反应中被非常有效和准确地修复，利用同一细胞中存在的第二个完整染色体副本的遗传信息。我们将使用这个DNA错误折叠和切割系统(以及另一个使用分子剪刀的系统)在特定的染色体位置产生可修复的DNA断裂，并研究它们通过同源重组进行的修复。我们将集中讨论活细胞中DNA断裂的修复机制。这是最不为人所知的领域，也是我们的研究能够产生最大影响的领域。多年的遗传学研究已经确定了与同源重组有关的基因，并在试管中对它们编码的蛋白质进行了研究。然而，在活细胞中研究这种反应的实验系统和进行这种分析的方法最近已经发展到可以取得实质性进展的阶段。使用显微镜，我们将实际观察修复反应，因为它们发生在活细胞中。我们还将分离和分析“在修复断裂的过程中捕获”的DNA分子。最后，我们将观察DNA断裂修复对染色体复制及其在下一代细胞中分布的间接影响。我们用于制造DNA断裂并分析其修复的复杂系统，与大肠杆菌作为实验有机体的丰富知识及其内在优势(如小体积和快速生长速度)将被用来取得比更复杂的生物体更快的进展。这项工作不仅将阐明活的大肠杆菌(一种重要的细菌病原体)中DNA断裂实际上是如何修复的，而且它将是在人类细胞中实施类似实验策略的第一步。一个例子将有助于说明这一点。我们打算第一次从染色体上的单个DNA修复位点分离DNA，并用电子显微镜分析这一DNA。即使对于460万个碱基对的染色体来说，这也不是微不足道的。然而，如果我们成功了，我们将开创在人类细胞中实现DNA类似目标的先河，即必须从超过40亿个碱基对的DNA中分离出特定的修复事件。