Biophysical and Structural Analysis of Recombination Repair Proteins

我们的基因组物质，DNA，不断受到环境中的物质（辐射，污染），我们从饮食中摄取的化学物质的攻击，它也表现出固有的不稳定性，因为它在复制，转录或在细胞分裂期间分裂。事实上，据估计，一个人类细胞每天会遭受7万多次单链断裂或碱基损伤，以及大约20次双链断裂。如果没有有效的修复，这些DNA损伤将会累积并导致诸如癌症或进行性神经退行性疾病等疾病。为了应对如此高水平的损伤，我们的细胞因此配备了许多DNA修复机制，每种机制都专门针对和消除不同类型的病变。然而，许多个体携带的可遗传突变会影响这些DNA修复途径的效率。例如，BRCA1、BRCA2、PALB2或RAD51副基因突变的个体易患乳腺癌和卵巢癌，这些癌症的发生频率很高。相同基因的突变也可能导致范可尼贫血，这是一种遗传性疾病，其特征是先天性异常，进行性骨髓衰竭，易患头颈癌和血癌。这些基因编码的蛋白质促进DNA双链断裂的修复，这可能是最危险的损伤形式，因为它们的低效修复会导致DNA易位或部分染色体的丢失。本研究旨在通过对DNA双链断裂修复过程中一些关键因素的结构、生物物理和生化分析，为DNA双链断裂修复机制提供新的见解。特别是，我们将确定RAD52的近原子结构，无论是单独的还是与DNA结合的，以及由RAD51B、RAD51C、RAD51D和XRCC2（缩写为BCDX2）四种蛋白质组成的RAD51平行复合体的结构。这些因子在同源重组修复（促进DNA双链断裂修复的过程）中起着关键作用，对避免癌症也很重要。结构分析将得到机制分析（生物物理和生化）的支持，这将揭示它们的作用机制。总之，我们的研究将提供详细的见解，为什么这些重要修复因子的患者衍生突变导致人类疾病。这些蛋白质的结构将使用一种称为冷冻电子显微镜（cryo-EM）的最先进技术来确定。为了做到这一点，我们感兴趣的蛋白质将从所有其他细胞成分中纯化出来，然后冷冻在冰上，这样它们就可以被电子轰击，产生单个分子的显微镜图像。这些被用来重建分子的三维形状或结构。一旦我们知道了它们的结构，我们就可以开始理解为什么突变会损害它们的活性并导致人类疾病。弗朗西斯克里克研究所于2015年开业，是欧洲最大的生物医学研究机构，我们很幸运拥有最强大的显微镜之一，以及一流的技术支持，这将使我们能够将这些研究成果付诸实践。

Our genomic material, DNA, is continually attacked by agents in the environment (radiation, pollution), chemicals that we ingest in our diet, and it also shows an inherent instability as it is replicated, or transcribed, or divided during cell division. Indeed, it is estimated that a single human cell will suffer more than 70,000 single strand breaks or base damages, and around 20 double strand breaks, each day. Without efficient repair, these lesions in DNA will accumulate and lead to diseases such as cancer or progressive neurodegenerative disorders. To cope with such high levels of damage, our cells are therefore equipped with a number of DNA repair mechanisms, each of which is specialised to target and remove different types of lesions.However, many individuals carry inheritable mutations that affect the efficiency of these DNA repair pathways. For example, individuals with mutations in the BRCA1, BRCA2, PALB2, or RAD51 paralog genes, are predisposed to breast and ovarian cancers which occur with a high frequency. Mutations in the same genes can also cause Fanconi anemia, a genetic disorder characterised by congenital abnormalities, progressive bone marrow failure and predisposition to head, neck and blood cancers. These genes encode proteins that promote the repair of DNA double-strand breaks, which represent possibly the most dangerous form of damage, as their inefficient repair can lead to DNA translocations or loss of part of a chromosome. This proposal aims to provide new insights into the mechanisms of DNA double-strand break repair, through structural, biophysical and biochemical analysis of some of the key factors in the process. In particular, we will determine the near atomic structure of RAD52, both alone and bound to DNA, and also that of a RAD51 paralog complex composed of four proteins, RAD51B, RAD51C, RAD51D and XRCC2 (abbreviated to BCDX2). These factors are key players in homologous recombinational repair, the process that promotes the repair of DNA double strand breaks, and are important for cancer avoidance. The structural analyses will be supported by mechanistic analyses (biophysical and biochemical) that will shed new light into their mechanism of action. Together, our studies will provide detailed insights into why patient-derived mutations in these important repair factors lead to human disease. The structures of these proteins will be determined using a state-of-the-art technique called cryo-electron microscopy (cryo-EM). To do this, the proteins we are interested in will be purified away from all other cellular components, and then frozen in ice so that they can be bombarded with electrons to produce microscope images of individual molecules. These are used to reconstruct the 3D shape, or structure, of the molecule. Once we know their structure, we can start to understand why mutations compromise their activity and cause human disease. At the Francis Crick Institute, which opened in 2015 and represents the largest biomedical research facility in Europe, we are fortunate to have one of the most powerful microscopes, and superb technical backup, that will enable us to bring these studies to fruition.