Systematic Genetic Analysis of Yeast NHEJ

描述（由申请人提供）：DNA双链断裂（DSB）修复是基因组维持的中心过程，大致分为同源重组（HR）和非同源末端连接（NHEJ）途径。其中，直接连接DSB末端的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的准确修复的基因组看护者，强调了其在基因组稳定性中的二分作用。先前的工作已经导致了几乎所有真核NHEJ蛋白的明显鉴定。其中包括：（i）结构末端结合蛋白Ku;（ii）DNA连接酶IV，由其催化亚基（Lig 4/Dnl 4）和两种支持蛋白（XRCC 4/Lif 1和XLF/Nej 1）组成;和（iii）Pol X家族的末端加工聚合酶（Pol <，Pol ;/Pol 4）。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。目前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，目前对其部分如何在空间和时间上组装到限制性DSB底物上的了解甚少。一旦到达那里，NHEJ酶使用知之甚少的机制来克服在由不稳定缔合的一半组成的DNA底物上催化反应的独特挑战。该项目将在芽殖酵母模式生物中使用强大且新型的遗传分析来探索这些悬而未决的问题，其中有四个具体目标：（i）Ku和DNA连接酶IV之间的相互作用，这些相互作用招募并有效地定位连接酶进行催化;（ii）DNA连接酶IV BRCT结构域在支持NHEJ方面的特异性和多重功能;（iii）Pol X家族DNA聚合酶的特定特征，其仅允许它们在NHEJ期间催化某些合成事件;和（iv）DNA连接酶IV的催化的类似特定特征，其优化其连接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.