Systematic Genetic Analysis of Yeast NHEJ

描述(申请人提供)：DNA双链断裂(DSB)修复是基因组维护的核心过程，大致分为同源重组(HR)和非同源末端连接(NHEJ)途径。其中，DSB末端的直接连接NHEJ最有可能执行导致癌症的染色体重排，因为这种连接通常缺乏广泛的同源性。然而，NHEJ也是促进DSB准确修复的基因组看守者，这突显了它在基因组稳定中的二分性作用。以前的工作已经导致了几乎所有真核细胞NHEJ蛋白的明显鉴定。它们包括：(I)结构末端结合蛋白Ku；(Ii)DNA连接酶IV，由其催化亚基(Lig4/Dnl4)和两个支持蛋白(XRCC4/Lif1和XLF/Nej1)组成；以及(Iii)Pol X家族的末端加工聚合酶(Pol&lt；，Pol；/Pol4)。这些不同蛋白质的许多特征也是已知的，包括大量的结构信息。尚不清楚的是，它们如何相互作用，以及DNA如何实现修复的动态过程。在NHEJ期间使用了广泛的蛋白质结构，但目前对其部分如何在空间和时间上组装到有限的DSB底物上的了解很少。一旦有了这些，NHEJ酶使用鲜为人知的机制来克服在由不稳定相关的一半组成的DNA底物上催化反应的独特挑战。这个项目将在萌芽中的酵母模式生物中使用强大而新颖的遗传分析来探索这些悬而未决的问题，有四个具体的目标：(I)Ku和DNA连接酶IV之间的相互作用，它招募并有效地定位用于催化的连接酶；(Ii)DNA连接酶IV BRCT结构域在支持NHEJ中的特定和多重功能；(Iii)Pol X家族DNA聚合酶的特殊功能，它只允许它们催化NHEJ中的某些合成事件；以及(Iv)DNA连接酶IV催化的类似的特定功能，它优化了它连接DSB末端的能力。

DESCRIPTION (provided by applicant): DNA double-strand break (DSB) repair is a central process in genome maintenance, broadly divided into homologous recombination (HR) and nonhomologous end joining (NHEJ) pathways. Of these, NHEJ, the direct ligation of DSB ends, is most likely to execute the chromosomal rearrangements that cause cancer because such junctions typically lack extensive homology. NHEJ is also a genome caretaker that promotes accurate repair of DSBs, however, underscoring its dichotomous role in genome (in)stability. Prior work has led to the apparent identification of nearly all eukaryotic NHEJ proteins. These include: (i) the structural end- binding protein Ku; (ii) DNA ligase IV, comprised of its catalytic subunit (Lig4/Dnl4) and two supporting proteins (XRCC4/Lif1 and XLF/Nej1); and (iii) end processing polymerases of the Pol X family (Pol <, Pol ;/Pol4). Many features of these various proteins are also known, including substantial structural information. What is not known is how they interact with each other and the DNA to achieve the dynamic process of repair. There is an extensive protein architecture used during NHEJ with currently little insight into how its parts assemble onto the limiting DSB substrate in both space and time. Once there, NHEJ enzymes use poorly understood mechanisms to overcome the unique challenge of catalyzing reactions on a DNA substrate comprised of unstably associated halves. This project will explore these outstanding issues using powerful and novel genetic assays in the budding yeast model organism, with four specific aims addressing: (i) the interactions between Ku and DNA ligase IV that recruit and productively position the ligase for catalysis; (ii) the specific and multiple functions of the DNA ligase IV BRCT domains in supporting NHEJ; (iii) the specific features of Pol X family DNA polymerases that allow only them to catalyze certain synthetic events during NHEJ; and (iv) similar specific features of catalysis by DNA ligase IV that optimize its ability to join DSB ends.