Determining the role of Blm and Marcal1 helicases in replication fork remodeling and progression

描述(申请人提供)：细胞利用各种途径和信号级联来修复DNA损伤；然而，当DNA活跃复制时，基因组是如何修复的还不完全清楚。复制过程中的DNA修复有助于产生化疗耐药，而复制过程中受损的修复是许多罕见遗传病的原因。复制分叉重塑是复制过程中损伤相关修复的关键中间产物，然而，哪些蛋白质负责分叉回归和随后分解退化的中间产物以促进重新启动仍是未知的。这个项目将使用经典的果蝇遗传学和现代分子技术来测试两个候选解旋酶Marcal1和BLm在复制叉子重塑(修复的关键中间体)中的作用和冗余。BLM解旋酶在双链断裂的同源重组修复中具有重要作用。初步数据表明，BLm在复制相关功能和HR角色方面可能受到不同的调节；这可能影响TOP3α相互作用，这 是人力资源职能所必需的。这一假设将通过使用标准技术在复制中表征BLm-TOP3α结合来验证。最后，BLM还参与了通过重复区域促进复制进程。这一新的作用将在果蝇胚胎中进行创新分析，并结合分子技术进行测试。该项目的综合方法旨在澄清由于修复途径和蛋白质的部分冗余而产生的复杂数据解释。该项目将深入了解复制和修复途径如何共同工作，以保持基因组的稳定性，并有可能影响罕见疾病的治疗以及化疗设计。

DESCRIPTION (provided by applicant): The cell utilizes a variety of pathways and signaling cascades to repair DNA damage; however, how the genome is repaired when DNA is actively replicating is imperfectly understood. DNA repair during replication contributes to chemotherapeutic resistance while compromised repair during replication is responsible for many rare genetic diseases. Replication fork remodeling is a key intermediate in damage-associated repair during replication, however which proteins are responsible for fork regression and subsequent resolution of regressed intermediates to facilitate restart remains unknown. This project will test the roles and redundancy of two candidate helicases, Marcal1 and Blm, in replication fork remodeling (a key intermediate in repair) using classical Drosophila genetics as well as modern molecular techniques. Blm helicase has functions in homologous recombination repair of double-strand breaks. Preliminary data suggests that Blm may be differentially regulated in replication-related functions vs. HR roles; this may affect Top3α interactions, which are necessary for HR functions. This hypothesis will be tested through characterization of Blm-Top3α binding in replication using standard techniques. Lastly, Blm has also been implicated in facilitating replication progression through repetitive regions. This novel role will be tested usig innovative assays in Drosophila embryos in conjunction with molecular techniques. The combined approach of this project is designed to clarify complex data interpretation resulting from partial redundancy of repair pathways and proteins. This project will provide insight into how replication and repair pathways work together to preserve genomic stability with the potential to impact rare disease treatment as well as chemotherapeutic design.