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

在转录过程中，通过阻止RNA聚合酶产生的mRNA-DNA r环对DNA复制构成了相当大的障碍，当基因组复制时，需要清除mRNA-DNA r环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（m6A）的r环倾向于在G2/M期积累，并在细胞周期的G0/G1期耗尽。此外，他们还发现m6a结合蛋白YTHDF2也与r -环相关，敲除YTHDF2会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断裂的积累。索尔塔纳斯和博尔特团队一直在合作研究人类DNA修复解旋酶HelQ。HelQ是一种重要的DNA修复蛋白，HelQ基因缺失的多重影响突出显示，包括突变率升高、癌症发病率增加和不育。遗传分析表明，当DNA复制被修复时，HelQ可以控制和限制同源重组，从而在不发生基因重排的情况下恢复复制。但HelQ解旋酶如何参与这些事件的生化机制尚不清楚。HelQ是一种依赖于atp的DNA解旋酶，可将3‘至5’极性的单链DNA （ssDNA）易位。最近，在Soultanas和Bolt实验室的合作研究中，他们发现了一个令人兴奋的发现，即HelQ似乎定位于r环，这表明HelQ可能也参与了r环的处理和去除，从而为不受阻碍的复制扫清了道路。我们假设，在细胞周期的G2/M期，由于转录停滞而产生的有问题的r环在G0/G1期被标记为清除，从而清除了可能在S期干扰DNA复制的基因组障碍。HelQ在这些r环上被招募来解开它们并清除它们。这个项目的主要目的是提供体内和体外的证据来支持我们的假设。1. 我们将对适当的细胞系、野生型和HelQ敲除进行免疫染色实验，以确认并提供更多的体内数据，证明HelQ共定位于r -环和m6a标记的r -环2。我们将对WT和HELQ KO细胞进行m6A免疫染色，分别在不存在RNase、仅存在RNase A和同时存在RNase A +H的情况下进行。这可能使我们能够辨别m6A修饰的r环的分布和水平是否在HELQ耗尽后发生改变。3. 我们将使用gammaH2AX双链标记进行免疫染色实验，以评估HelQ的缺失是否会导致DNA损伤增加。4. 我们将进行补体实验来评估HelQ的表达是否能拯救HelQ-缺失的细胞。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. 我们将开展bio-ID实验来识别helq -互作物。

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.