DNA repair by alternative end-joining in Drosophila

当DNA发生断裂时，细胞会激活修复机制，将末端连接在一起。通常情况下，DNA修复是一个精确的过程，用来保护基因组的完整性。然而，令人惊讶的是，用于连接断裂DNA末端的某些类型的修复非常容易出错。目前尚不清楚这种情况是如何发生的，以及为什么会发生。这项研究试图发现是什么使末端连接修复如此容易出错，以及哪些DNA序列背景可能有利于这种不精确。这些结果将提供关键信息，对于那些想要故意防止或促进不准确修复的研究人员来说，例如在进行基因组编辑操作时，这些信息可能被证明是有用的。此外，这项研究将为本科生和研究生水平的科学家提供分子生物学、基因组学、计算建模和有效指导技术方面的可移植技能。最后，该项目将聘请一个由现任和前任实验室成员组成的网络，作为基础科学研究的公共大使。替代的末端连接是DNA双链断裂修复的一种容易出错的机制，这会导致断裂部位的小片段缺失和插入。越来越多的证据表明，这些DNA变化可能是专业的跨病变DNA聚合酶theta与各种辅助蛋白迭代作用的结果。然而，人们对这些蛋白质如何与断裂附近的DNA序列相互作用以及DNA序列本身可能发挥的作用知之甚少。一种假设是，断裂一侧的DNA形成了短暂的二级结构，如短环和发夹，这些结构推动了新单链DNA的产生。然后，新生的链与断裂另一侧的微同源序列配对，作为完成修复的模板，从而产生各种修复产品。这一假说将在果蝇--黑腹果蝇身上得到验证。果蝇是一种生物，它严重依赖另一种末端连接修复方式，特别适合研究不同的组织类型或发育阶段如何影响这一容易出错的过程。遗传操作、高通量扩增子测序和计算模型的系统组合将被用来表征不同类型的二级结构形成序列以及它们作为替代末端连接的结果产生的各种类型的修复连接。最后，将评估替代末端连接在修复由基因组编辑工具CRISPR-Cas9引起的断裂中的可能作用；这一实验的结果可能提供一种途径，将对这种类型的DNA修复的基本理解扩展到改善目前的基因组编辑做法。

When breaks occur in DNA, cells activate repair mechanisms to join the ends back together. Usually DNA repair is a precise process that serves to preserve the integrity of the genome. Surprisingly though, some types of repair used for joining broken DNA ends are very prone to errors. How and why this occurs is not clear. This research seeks to discover what makes end-joining repair so error-prone and what DNA sequence contexts might favor such imprecision. The results will provide crucial information that could prove useful for researchers who want to intentionally prevent or promote inaccurate repair, such as when conducting genome editing manipulations. Furthermore, the research will provide undergraduate and graduate-level scientists with transferable skills in molecular biology, genomics, computational modeling, and effective mentoring techniques. Finally, the project will engage a network of current and former lab members as public ambassadors for basic science research.Alternative end-joining is an error-prone mechanism of DNA double-strand break repair, which leads to small deletions and insertions at the break site. Accumulating evidence suggests that these DNA changes may result from the iterative action of the specialized translesion DNA polymerase, theta, with various accessory proteins. However, little is known about how these proteins interact with DNA sequences in the vicinity of the breaks and what role the DNA sequences themselves might play. One hypothesis is that the DNA on one side of the break forms transient secondary structures, such as short loops and hairpins, which drive production of new single-stranded DNA. The nascent strands then pair with microhomologous sequences on the other side of the break and serve as templates for completing the repair, thereby leading to a variety of repair products. This hypothesis will be tested in the fruit fly, Drosophila melanogaster, an organism that relies heavily on alternative end-joining repair and is uniquely amenable to studying how different tissue types or developmental stages might influence this error-prone process. A systematic combination of genetic manipulation, high throughput amplicon sequencing, and computational modeling will be used to characterize different types of secondary-structure forming sequences and the various classes of repair junctions they produce as a result of alternative end-joining. Finally, a possible role of alternative end-joining in repair of breaks induced by the genome editing tool, CRISPR-Cas9, will be assessed; the results of this experiment could provide an avenue for extending the basic understanding of this type of DNA repair to improving current practices for genome editing.