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

DNA对所有生物体都至关重要，因为它编码生长，生存和遗传所需的信息。染色体在非常长的DNA分子中包含这种遗传信息（通常长度超过数百万个分子构建块，称为碱基对）。这个巨大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受损的DNA。染色体中一个未修复的DNA断裂会导致细胞死亡。断裂不仅需要有效地修复，而且还必须准确地修复。不准确的修复会导致导致癌症或遗传疾病的遗传改变。此外，DNA断裂在放疗和化疗等治疗中用于杀死癌细胞。断裂修复途径也为新抗生素的开发提供了靶点。了解DNA分子如何断裂以及如何修复对于未来的临床实践至关重要。有趣的是，并非所有的DNA序列都同样容易断裂。由于它们的反向重复性质，回文DNA序列可以在染色体中错误折叠，并且这些错误折叠的结构具有较高的断裂概率。已知DNA回文是染色体中与胎儿中的人类遗传疾病和成人中的癌症相关的位置。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E.我们发现一种称为SbcCD的特定蛋白质（也存在于人类中，称为Rad 50/Mre 11）负责在回文序列处产生DNA断裂。然而，这些断裂在称为同源重组的反应中非常有效和准确地修复，使用相同细胞中存在的染色体的第二个未断裂拷贝上的遗传信息。我们将使用这种DNA错误折叠和切割系统（以及另一种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，并通过同源重组研究其修复。我们将专注于活细胞中DNA断裂的修复机制。这是我们所知最少的领域，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参与同源重组的基因，并且已经在试管中研究了它们编码的蛋白质。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被捕获”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的分布的间接影响。大肠杆菌作为一种实验生物，其固有的优点（如体积小，生长速度快）将被用来取得更快的进展比它可能与更复杂的生物。这项工作不仅将阐明DNA断裂是如何在活的大肠杆菌中修复的。大肠杆菌（一种重要的细菌病原体），但这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。我们打算首次从染色体上的单个DNA修复位点分离DNA，并通过电子显微镜分析该DNA。即使对于长度为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，我们将为人类细胞中DNA的类似目标开辟道路，其中必须从超过40亿个DNA碱基对中分离出特定的修复事件。

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.