The human DNA helicase HelQ is recruited at R-loops and helps in their resolution and repair.

人类 DNA 解旋酶 HelQ 在 R 环上被招募，有助于其解析和修复。

• 批准号: 2434496
• 金额: --
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2434496
• 关键词: human; DNA; helicase; HelQ; recruited

Introduction mRNA-DNA R-loops created during transcription by stalling RNA polymerases pose a considerable obstacle to DNA replication and need to be cleared off for a smooth progression into the S phase of the cell cycle when the genome is replicated. Otherwise, the accumulation of R-loops can lead to genome instability. In a recent paper, Ruzov and collaborators discovered that N6-methyladenosine (m6A)-containing R-loops tend to accumulate during the G2/M phase and are depleted during the G0/G1 phase of the cell cycle. Furthermore, they showed that the m6A-binding protein YTHDF2 also associates with R-loops and a ydhdf2 knockout leads to increased R-loops levels, cell growth retardation and accumulation of double strand DNA breaks in mammalian cells. The Soultanas and Bolt groups have been working collaboratively with a human DNA repair helicase known as HelQ. HelQ is an important DNA repair protein, highlighted by multiple effects of helq gene loss, including elevated mutation rates, increased cancer incidence and sterility. Genetic analyses have led to models implicating HelQ in controlling and limiting homologous recombination when DNA replication is being repaired, so that replication can resume without genetic rearrangements occurring. But biochemical mechanisms for how HelQ helicase contributes to these events are unknown. HelQ is an ATP-dependent DNA helicase that translocates single stranded DNA (ssDNA) with 3' to 5' polarity. Recent collaborative work in the Soultanas and Bolt labs has resulted in the exciting discovery that HelQ appears to localize at R-loops, suggesting that HelQ may also be involved in processing and removing R-loops, clearing the way for replication to occur unimpeded. Hypothesis We hypothesize that problematic R-loops created by stalled transcription during the G2/M phase of the cell cycle are marked for removal during the G0/G1 clearing the genome from obstacles that might interfere with DNA replication during the S phase. HelQ is recruited at these R-loops to unwind them and clear them off. Project The main aim of this project is to provide in vivo and in vitro evidence in support of our hypothesis. 1. We will carry out immunostaining experiments with appropriate cell lines, wildtype and helq-knockout, to confirm and provide additional in vivo data that HelQ co-localizes at R-loops and also at m6A-marked R-loops 2. We will carry out immunostaining of m6A on WT and HELQ KO cells in the presence of no RNases, in the presence of RNase A only, and in the presence of both RNase A +H. This may allow us to discern whether the distribution and levels of m6A modified R-loops is altered following HELQ depletion. 3. We will use the gammaH2AX double strand marker to carry out immunostaining experiments to assess whether depletion of HelQ leads to increased DNA damage. 4. We will carry out complementation experiments to assess whether expression of HelQ rescues HelQ-depleted cells. 5. We will investigate whether HelQ co-immunoprecipitates with RNA polymerase. 6. We will express/purify the human HelQ protein and use different synthetic oligonucleotide substrates to study its activities. 7. We will carry out bio-ID experiments to identify HelQ-interactants.

在转录过程中通过停止RNA聚合酶而产生的mRNA-DNA R环对DNA复制构成相当大的障碍，并且需要在基因组复制时清除以顺利进入细胞周期的S期。否则，R环的积累会导致基因组不稳定。在最近的一篇论文中，Ruzov和合作者发现，含有N6-甲基腺苷（m6 A）的R环倾向于在细胞周期的G2/M期积累，并在G 0/G1期耗尽。此外，他们表明m6 A结合蛋白YTHDF 2也与R环相关，并且ydhdf 2敲除导致哺乳动物细胞中R环水平增加，细胞生长迟缓和双链DNA断裂积累。Soultanas和Bolt团队一直在与一种名为HelQ的人类DNA修复解旋酶合作。HelQ是一种重要的DNA修复蛋白，helq基因缺失的多重影响，包括突变率升高，癌症发病率增加和不育。遗传分析已经产生了涉及HelQ在DNA复制被修复时控制和限制同源重组的模型，使得复制可以在不发生基因重排的情况下恢复。但HelQ解旋酶如何促成这些事件的生化机制尚不清楚。HelQ是ATP依赖性DNA解旋酶，其以3'至5'极性易位单链DNA（ssDNA）。Soultanas和Bolt实验室最近的合作工作导致了令人兴奋的发现，即HelQ似乎定位于R环，这表明HelQ也可能参与处理和去除R环，为复制畅通无阻地发生扫清了道路。假设我们假设，在细胞周期的G2/M期期间由停滞的转录产生的有问题的R环被标记为在G 0/G1期间清除基因组中可能干扰S期期间DNA复制的障碍。HelQ在这些R环处被招募来解开它们并清除它们。本项目的主要目的是提供体内和体外证据来支持我们的假设。1.我们将用适当的细胞系、野生型和helq敲除进行免疫染色实验，以确认并提供HelQ共定位于R环以及m6 A标记的R环2的额外体内数据。我们将在不存在RNA酶、仅存在RNA酶A以及存在RNA酶A +H的情况下，对WT和HELQ KO细胞进行m6 A免疫染色。这可以使我们辨别在HELQ耗尽后m6 A修饰的R环的分布和水平是否改变。3.我们将使用γ H2 AX双链标记进行免疫染色实验，以评估HelQ的缺失是否导致DNA损伤增加。4.我们将进行互补实验以评估HelQ的表达是否拯救HelQ耗尽的细胞。5.我们将研究HelQ是否与RNA聚合酶共免疫沉淀。6.我们将表达/纯化人HelQ蛋白，并使用不同的合成寡核苷酸底物来研究其活性。7.我们将进行bio-ID实验以鉴定HelQ相互作用物。