Double-strand break repair and suppression of recombination in D. melanogaster

描述(申请人提供)：我们的基因组经历了大量的DNA损伤，来自正常细胞过程的外源和内源副产品。细胞识别和修复DNA损伤的能力对于维持基因组的完整性至关重要。DNA修复失败可能导致突变、细胞死亡、过早衰老和癌症。这里提出的工作旨在分析一种特定的和高度有害的DNA损伤类型-双链断裂(DSB)的修复，并在遗传易驯化的有机体的背景下确定修复DSB的机制。DSB可以通过非同源末端连接或同源重组来修复，其中末端被修饰和连接，或者同源重组，其中使用相同的序列(例如同源或姐妹染色单体)作为供体模板来修复DSB并恢复在断裂时丢失的信息。正常细胞对供体模板的同源性高度敏感，因为分歧序列之间的重组修复受到高度抑制，以避免基因组重排和不稳定。然而，负责这种抑制的遗传成分并没有被清楚地描绘出来。最后，RecQ解旋酶是一个蛋白质家族，在解开DNA底物和维持基因组完整性(包括抑制不同序列之间的重组)方面具有独特和重叠的作用。在DSB修复的背景下，这些家庭成员在物种内部和跨物种之间的冗余的可能性尚未得到解决。通过对果蝇的分子和遗传分析，两种新的DSB修复方法将被用来1)确定简单DSB在整个有机体中被修复的机制，2)描述分歧序列之间的重组是如何被抑制的，以及3)建立RecQ解旋酶在物种内和物种之间的功能冗余。重要的是，为了维持区域奖励机制的目标，该项目将为本科生研究人员提供广泛的分子和基因技术实践经验以及假说驱动的培训，这将为未来在生物医学研究和/或与健康相关的职业生涯提供宝贵的技能集。

DESCRIPTION (provided by applicant): Our genomes experience a large quantity of DNA damage, from both exogenous sources and endogenous byproducts of normal cellular processes. The ability of the cell to recognize and repair DNA damage is essential for maintenance of genomic integrity. Failures in DNA repair can lead to mutations, cell death, premature aging, and cancer. The work proposed here aims to analyze repair of a specific and highly deleterious type of DNA damage, a double-strand break (DSB), and to determine the mechanism by which DSBs are repaired in the context of a genetically tractable organism. DSBs are repaired by either non- homologous end joining, where the ends are modified and joined, or homologous recombination, where identical sequences (such as a homolog or sister chromatid) are used to as a donor template to repair the DSB and restore information lost at the break. Normal cells are highly sensitive to homology of the donor template, as recombination repair between diverged sequences is highly suppressed to avoid genome rearrangements and instability. However, the genetic components responsible for this suppression are not clearly delineated. Lastly, RecQ helicases are a family of proteins that have unique and overlapping roles in unwinding DNA substrates and maintaining genome integrity (including suppressing recombination between diverged sequences). The potential for redundancy of these family members within and across species has not been addressed in the context of DSB repair. Using molecular and genetic analyses in Drosophila melanogaster, two novel DSB repair assays will be utilized to 1) determine the mechanisms by which simple DSBs are repaired in the context of a whole organism, 2) delineate how recombination between diverged sequences is suppressed, and 3) establish functional redundancy of RecQ helicases within and across species. Importantly, to maintain goals of the AREA award mechanism, this project will give undergraduate researchers hands-on experience in a wide variety of molecular and genetic techniques and hypothesis-driven training, which will provide a valuable skill set for a future career in biomedical research and/or health-related careers.