DNA damage tolerance pathway choice in Drosophila

果蝇DNA损伤耐受途径的选择 项目摘要 当复制叉遇到DNA损伤时，它们必须绕过这些障碍， DNA损伤耐受机制或DNA合成将停止，导致基因组中的缺口， 基因组的不稳定性和最终的细胞死亡。细胞用于旁路的策略包括 易出错的跨损伤合成聚合酶的募集， 复制叉，并在损伤下游再引发，然后间隙填充。的选择 损伤耐受机制是重要的，因为一些途径比其他途径更具致突变性。 然而，在多细胞动物中病变旁路选择的控制还没有很好地理解， 特别是在不同组织的情况下。这些知识对于理解 正常细胞和癌细胞如何处理由以下原因引起的复制阻断病变 用化疗剂治疗。 我们最近发现REV1蛋白是DNA损伤耐受性的关键介质 在果蝇中。REV1促进跨病变聚合酶的募集以绕过受损的 碱基，我们已经证明这是快速分裂组织的首选耐受机制， 发展中的苍蝇此外，REV1似乎通过一个介导的模板转换， 未知机制在这里描述的实验中，我们将使用域特异性突变体， 遗传分析和幼虫成虫盘中的细胞测定，以表征REV 1 协调各种损伤耐受途径以促进DNA复制的持续。在 此外，我们将使用遗传和生物化学方法来鉴定参与 损伤容限我们的调查将得到我们开发的新技术的帮助 评估细胞和组织中的DNA修复和诱变， 修复和复制突变体。在我们的实验设计中使用整个果蝇， 我们有机会在组织特异性的背景下研究损伤耐受性， 发展总之，我们提出的研究将推进我们的长期目标， 为什么不同的DNA损伤耐受机制优先用于不同的环境 以及这种旁路策略的选择如何影响细胞存活和基因组稳定性。

DNA damage tolerance pathway choice in Drosophila Project Summary When replication forks encounter DNA lesions, they must bypass these roadblocks through DNA damage tolerance mechanisms or DNA synthesis will stop, leading to gaps in the genome, genomic instability, and eventual cell death. Strategies that cells use for bypass include the recruitment of error-prone translesion synthesis polymerases, template switching at the replication fork, and repriming downstream of the lesion followed by gap filling. The choice of damage tolerance mechanism is important, as some pathways are more mutagenic than others. However, the control of lesion bypass pathway choice in metazoans is not well understood, particularly in the context of different tissues. Such knowledge is critical in order to understand how both normal and cancerous cells deal with replication-blocking lesions that result from treatment with chemotherapeutic agents. We have recently discovered that the REV1 protein is a key mediator of DNA damage tolerance in Drosophila. REV1 promotes the recruitment of translesion polymerases to bypass damaged bases, which we have shown is the preferred tolerance mechanism in rapidly dividing tissues in the developing fly. In addition, REV1 appears to mediate template switching through an unknown mechanism. In the experiments described here, we will use domain-specific mutants, genetic analysis, and cellular assays in larval imaginal discs to characterize how REV1 coordinates various damage tolerance pathways to promote continuance of DNA replication. In addition, we will use both genetic and biochemical methods to identify new proteins involved in damage tolerance. Our investigations will be aided by novel techniques that we have developed to assess DNA repair and mutagenesis in both cells and tissues and by a rich collection of DNA repair and replication mutants. The use of whole Drosophila in our experimental design provides us with an opportunity to study damage tolerance in the context of tissue specificity and development. Together, our proposed studies will advance our long-term goal to understand why different DNA damage tolerance mechanisms are preferentially used in different contexts and how this choice of bypass strategy impacts cellular survival and genome stability.