Structural and functional studies of DNA mismatch repair proteins

DNA错配修复蛋白的结构和功能研究

• 批准号: RGPIN-2017-05828
• 负责人: Guarne, Alba
• 金额: $ 4.37万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=652577
• 关键词: Structural; functional; studies; DNA; mismatch

The integrity of DNA is constantly threatened by damaging insults that can lead to chromosomal rearrangements or cell death. The long-term goal of our research program is to understand how DNA damage is efficiently repaired, in turn preventing genome instability. One of the most fascinating aspects of the DNA damage response is how repair proteins find the lesions regardless of context, thereby ensuring the repair of each and every lesion. Our laboratory studies how the proteins that cut DNA based on its shape (known as structure-specific nucleases) recognize and cut their target DNA. Our overarching hypothesis is that structure-specific nucleases use common DNA binding mechanisms, but achieve specificity through their interaction with pathway-specific factors. To probe this idea, we will use a combination of biochemical, biophysical and structural biology techniques to study how structure-specific nucleases in two DNA repair pathways bind and cut DNA. DNA mismatch repair corrects errors that occur while DNA is being copied. Two conserved proteins are essential for this process: MutS and MutL. Over the years, my laboratory has made key contributions to the functions of MutL. However, MutL does not look like any other nuclease and, therefore, we do not understand how it cuts DNA yet. In the next five years, we will address this question by determining the 3-dimensional structure of MutL bound to DNA. Leveraging our experience with MutL, we have recently broaden the scope of our research program to characterize structure-specific nucleases in other repair pathways. Breaks affecting both DNA strands are the most dangerous form of damage because the broken pieces do not retain information of their context. Single strand-annealing repairs double-strand breaks between adjacent repeat sequences. Although this repair mechanism causes the loss of genetic information, it is prevalent in humans, due to the high frequency of homologous repeat sequences present in our genome. We will determine how the adaptor protein Saw1 recruits the structure-specific nuclease Rad1-Rad10 to repair intermediates. This is important because Rad1-Rad10 can process several types of damage and, therefore, the adaptor proteins determine the lesion to be processed. Our work will provide fundamental mechanistic insights into the roles of these two nucleases in DNA repair and genome maintenance. The latter is specially important for healthy aging. Canada has been described as a “greying nation” because it has more people over 65 than under 15, underscoring the indirect benefits of our research.

DNA的完整性不断受到可导致染色体重排或细胞死亡的破坏性损害的威胁。我们研究项目的长期目标是了解DNA损伤是如何有效修复的，从而防止基因组不稳定。DNA损伤反应最吸引人的一个方面是修复蛋白如何找到病变，从而确保每个病变的修复。我们的实验室研究根据形状切割DNA的蛋白质（称为结构特异性核酸酶）如何识别和切割目标DNA。我们的总体假设是，结构特异性核酸酶使用常见的DNA结合机制，但通过与途径特异性因子的相互作用实现特异性。为了探究这一观点，我们将结合生物化学、生物物理和结构生物学技术来研究两种DNA修复途径中结构特异性核酸酶如何结合和切割DNA。DNA错配修复可以纠正DNA复制过程中出现的错误。两个保守蛋白在这个过程中是必不可少的：MutS和MutL。多年来，我的实验室对MutL的功能做出了重要贡献。然而，MutL看起来不像任何其他核酸酶，因此，我们还不知道它是如何切割DNA的。在接下来的五年里，我们将通过确定与DNA结合的MutL的三维结构来解决这个问题。利用我们在MutL方面的经验，我们最近扩大了我们研究项目的范围，以表征其他修复途径中的结构特异性核酸酶。影响两条DNA链的断裂是最危险的损伤形式，因为断裂的片段不能保留其环境的信息。单链退火修复相邻重复序列之间的双链断裂。尽管这种修复机制会导致遗传信息的丢失，但由于我们基因组中存在高频率的同源重复序列，它在人类中很普遍。我们将确定接头蛋白Saw1如何招募结构特异性核酸酶Rad1-Rad10来修复中间产物。这很重要，因为Rad1-Rad10可以处理几种类型的损伤，因此，接头蛋白决定要处理的病变。我们的工作将为这两种核酸酶在DNA修复和基因组维持中的作用提供基本的机制见解。后者对于健康老龄化尤为重要。加拿大被称为“老龄化国家”，因为65岁以上的人比15岁以下的人多，这凸显了我们研究的间接好处。