Investigating novel mechanisms of genetic stability at the replication fork

人体是由数万亿个细胞组成的。为了补充老的或垂死的细胞，每天必须通过祖细胞的复制和分化产生数十亿个新细胞。为了做到这一点，我们的细胞必须小心地复制它们的遗传物质，DNA，并正确地将其传递给它们的子细胞。这项具有挑战性的任务是由几种蛋白质完成的，包括解除DNA的解旋酶和一系列从亲本链复制DNA的称为DNA聚合酶的蛋白质。这个机器的核心是DNA聚合酶Epsilon (PolE)，它是解绕DNA解旋酶的一个组成部分，也合成部分DNA。DNA复制功能障碍会导致DNA中错误的积累，从而导致遗传疾病和癌症。因此，了解极点如何工作并发现负责高效和准确的DNA复制的机制对人类健康以及癌症预防和治疗至关重要。我们的身体有200多种不同类型的细胞。负责产生如此不同的细胞库的过程被称为细胞分化，它取决于特定基因集的表达。因此，尽管一个人的所有细胞共享相同的遗传物质，但在细胞中只有这些基因的一部分是活跃的。我们的基因被赋予一种叫做染色质的蛋白质结构，它主要由组蛋白组成，可以被修改以允许特定基因的表达。总的来说，决定我们基因功能的特定组蛋白和DNA修饰被称为表观基因组。重要的是，当细胞复制它们的遗传物质时，包含在组蛋白及其修饰中的表观遗传信息也必须被复制。因此，这两个过程是不可逆转地交织在一起的。复制DNA的机制是如何将这两个过程结合在一起的，目前还不清楚。在我的博士后研究中，我发现PolE的两个组成部分POLE3和POLE4对于促进整个PolE蛋白复合物的稳定性及其在DNA复制过程中的功能至关重要。引人注目的是，我还发现这些蛋白质在DNA复制过程中与组蛋白结合，这表明它们可能参与了我们表观遗传信息的复制过程。这一令人兴奋的发现引发了一系列的研究问题，我现在打算在我的实验室里解决这些问题。特别是，我想了解整个极点复合体是如何执行这两个过程的，以及它们对细胞功能的相对贡献。除此之外，最近的研究发现，POLE3和POLE4的缺失会增加癌细胞对某些特定抗癌药物的反应。这种敏感性增加的原因仍有待揭示，查明这一原因是我的建议的另一个根本目的。事实上，这些结果在为这些药物的作用机制提供重要线索的同时，也将确定POLE3和POLE4的主要功能，并确定潜在的新的治疗靶点和预测对这些药物敏感性的标记物。总之，回答这些令人兴奋的新问题将大大增加我们对遗传和表观遗传稳定性所需的基本过程的理解，并将有助于确定有效的癌症治疗方法。

The human body is made up of trillions of cells. To replenish old or dying cells, billions of new cells have to be generated each day by duplication and differentiation of progenitor cells. In order to do this, our cells have to carefully replicate their genetic material, the DNA, and correctly transmit it to their daughter cells. This challenging task is performed by several proteins, including helicases, that unwind DNA and a series of proteins called DNA polymerases that copy DNA from the parental strands. At the heart of this machinery is DNA Polymerase Epsilon (PolE) which is a component of the unwinding DNA helicase and also synthesizes part of the DNA. Dysfunction of DNA replication can lead to the accumulation of errors in the DNA and cause genetic diseases and cancer. Thus, understanding how PolE works and discovering the mechanism responsible for efficient and accurate DNA replication is essential for human health as well as for cancer prevention and therapy. Our body harbours more than 200 different cell types. The process that is responsible for the production of such a different repertoire of cells is called cellular differentiation and depends on the expression of specific sets of genes. Thus, despite all the cells of an individual share the same genetic material, only a subset of those genes are active in a cell. Our genes are endowed into a protein structure called chromatin, mainly composed of histone proteins, which can be modified to allow the expression of specific genes. Altogether, the repertoire of specific histones and DNA modifications that determine the functionality of our genes is known as the epigenome. Importantly, when cells duplicate their genetic material, the epigenetic information that is contained in histones and their modifications has also to be duplicated. Therefore, these two processes are irreversibly intertwined. How the machinery that replicates DNA couples these two processes remains poorly defined.During my post-doctoral studies, I discovered that two components of PolE, named POLE3 and POLE4, are important to promote the stability of the whole PolE protein complex and its functions during DNA replication. Strikingly, I also found that these proteins bind to histones during DNA replication, which suggests they might participate in the process of duplication of our epigenetic information. This exciting finding opened a series of research questions that I now aim to address in my laboratory. In particular, I want to understand how the whole PolE complex perform these two processes and their relative contributions to cellular function. In addition to this, recent studies have discovered that loss of POLE3 and POLE4 increases the response of cancer cells to some specific anti-cancer drugs. The reason for this increased sensitivity remains to be unveiled and its identification represents the other fundamental aim of my proposal. Indeed, these results while providing important clues in the mechanism of action of these drugs, will also determine the main functions of POLE3 and POLE4 and identify potential new therapeutic targets and markers predicting sensitivity to such drugs. In summary, answering these exciting new questions will significantly increase our understanding of essential processes required for genetic and epigenetic stability and will help identify effective cancer therapies.