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

核糖核酸聚合酶在转录过程中产生的m RNA-DNA R环对DNA复制构成相当大的障碍，当基因组被复制时，为了顺利进入细胞周期的S阶段，需要清除这些环。否则，R-环的积累可能会导致基因组不稳定。在最近的一篇论文中，Ruzov和他的合作者发现，含有N6-甲基腺苷(M6A)的R-环往往在G2/M期积累，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，ydhdf2基因敲除会导致R-环水平增加，细胞生长迟缓，并在哺乳动物细胞中积累双链DNA断裂。Soultanas和Bolt团队一直在合作研究一种名为Helq的人类DNA修复解旋酶。Helq是一种重要的DNA修复蛋白，其突出表现为Helq基因缺失的多种影响，包括突变率增加、癌症发病率增加和不孕不育。遗传分析导致了一些模型，这些模型暗示当DNA复制被修复时，Helq控制和限制同源重组，这样复制就可以恢复，而不会发生基因重排。但Helq解旋酶如何对这些事件起作用的生化机制尚不清楚。HELQ是一种依赖于ATP的DNA解旋酶，它将单链DNA(SsDNA)以3‘到5’的极性易位。Soultanas和Bolt实验室最近的合作工作导致了一个令人兴奋的发现，即Helq似乎定位于R环，这表明Helq也可能参与处理和移除R环，为顺利进行复制扫清了道路。假设我们假设，在细胞周期的G2/M阶段，由于转录停滞而产生的有问题的R-环被标记为在G0/G1期间移除，从而在S阶段清除了可能干扰DNA复制的障碍。Helq被招募到这些R环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.我们将用合适的野生型和Helq基因敲除的细胞系进行免疫染色实验，以确认和提供额外的体内数据，即Helq共定位于R环和m6A标记的R环2。我们将在不存在RNase的情况下，仅在RNaseA存在的情况下，以及在RNaseA+H存在的情况下，对WT和Helq KO细胞进行m6A的免疫染色。这可能使我们能够区分在Helq缺失后，m6A修饰的R环的分布和水平是否发生了变化。3.我们将使用GammaH_2AX双链标记进行免疫染色实验，以评估HELQ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的表达是否拯救了Helq缺失的细胞。5.我们将研究HELQ是否与RNA聚合酶共沉淀。6.我们将表达/纯化人Helq蛋白，并使用不同的人工合成寡核苷酸底物来研究其活性。7.我们将开展生物识别实验，以鉴定Helq相互作用物。

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.