Roles of the human UAP56 helicase in co-transcriptional R-loop resolution and genome maintenance

项目摘要 共转录R环是长的DNA-RNA杂合体，可以在RNA聚合酶II的转录过程中产生。 虽然R环具有重要的生理功能，如类别转换重组和促进细胞内的蛋白质合成。 转录终止，持久的R环代表了DNA损伤，复制应激和 基因组不稳定性R环分辨率的缺陷见于神经退行性疾病和癌症。细胞具有 进化出不同的策略来防止病理性R环的积累。其中一个机制依赖于 人类保守的THO复合物及其相关的共转录因子UAP 56解旋酶。然而，在这方面， UAP 56如何在R环预防中起作用的详细机制仍有待确定。 我们的初步数据表明，UAP 56的缺失导致R环积累和R环介导的细胞凋亡。 基因组不稳定性此外，纯化的UAP 56比RNA-RNA更善于解旋RNA-DNA杂合体 复式。它还在体外解离模型R环。基于这些发现，我们假设UAP 56参与了 并解离共转录R环结构以防止它们在细胞中积累。我们还假设 UAP 56的R环解离活性在转录过程中需要通过其结合 配偶体CITREF和CHTOP，两种RNA结合蛋白。本R15提案的总体目标是确定 UAP 56如何帮助消除有害的共转录R环，以及这种活性如何受其辅因子的调节 来维持基因组的稳定性在本项目中，我们将通过三个具体目标来实现我们的目标：（1）研究共同的， UAP 56在细胞中的转录R环去除和基因组维持功能;（2）确定R环 UAP 56在体外的分辨功能;（3）表征UAP 56在R-loop去除中的调节作用， 生理伴侣，CNOREF和CHTOP。在目标1中，我们将检查R-loop水平和DNA损伤， 使用一组细胞和生物化学方法，包括使用免疫荧光（IF）， S9.6单克隆抗体，DRIP-qPCR在一组靶基因中进行DNA-RNA杂合积累，亚硫酸氢盐 靶基因测序监测胞嘧啶的转化，DRIPc-seq在基因组规模上绘制R环， 彗星试验和γ H2 AX焦点形成。在目标2中，我们用高度纯化的UAP 56及其解旋酶死亡突变体， 将研究它与一系列RNA/DNA和其他核酸底物的关系，以及它的能力， 解离DNA-RNA杂合体和R环，包括基于质粒的生理学相关R环。在Aim中 3、我们将纯化UAP 56和CHTOP，并确定它们对UAP 56在DNA解旋中活性的影响。 重组生化系统和细胞中的RNA杂合体和R环解析。我们项目的成果 将阐明共转录R环加工和基因组保存的一般机制。我们 我希望我们的努力有助于开发新的战略，以防止致病性R环 形成和治疗由其积累引起的神经变性疾病和癌症。 !一个！

PROJECT SUMMARY Co-transcriptional R-loops are long DNA-RNA hybrids that can arise during transcription by RNA Polymerase II. While R-loops serve important physiological functions, such as class switch recombination and promotion of transcription termination, persistent R-loops represent a major source of DNA damage, replication stress and genome instability. Defects in R-loop resolution are seen in neurodegenerative diseases and cancer. Cells have evolved different strategies to prevent the accumulation of pathological R-loops. One such mechanism relies on the conserved THO complex and its associated co-transcriptional factor UAP56 helicase in humans. However, the detailed mechanism how UAP56 functions in R-loop prevention remains to be determined. Our preliminary data have shown that depletion of UAP56 results in R-loop accumulation and R-loop-mediated genome instability. In addition, purified UAP56 is more adept at unwinding RNA-DNA hybrids than RNA-RNA duplex. It also dissociates model R-loops in vitro. Based on these findings, we hypothesize that UAP56 engages and dissociates co-transcriptional R-loop structures to prevent their accumulation in cells. We also hypothesize that the R-loop dissociation activity of UAP56 needs to be strictly regulated during transcription by its binding partner ALYREF and CHTOP, two RNA binding proteins. The overall goal of this R15 proposal is to determine how UAP56 helps eliminate harmful, co-transcriptional R-loops, and how this activity is regulated by its cofactors to maintain genome stability. In this project, we will achieve our goal by three specific aims: (1) examine co- transcriptional R-loop removal and genome maintenance function of UAP56 in cells; (2) determine the R-loop resolution function of UAP56 in vitro; (3) characterize the regulation of UAP56 in R-loop removal by its physiological partner, ALYREF and CHTOP. In Aim 1, we will examine the R-loop levels and DNA damage in UAP56 depleted cells using a set of cellular and biochemical methods, including immunofluorescence (IF) using the S9.6 monoclonal antibody, DRIP-qPCR in a set of target genes for DNA-RNA hybrid accumulation, bisulfite sequencing of target genes monitoring conversion of cytosines, DRIPc-seq to map R-loops on the genomic scale, comet assay and γH2AX foci formation. In Aim 2, with highly purified UAP56 and its helicase dead mutants, we will investigate its association with a series of RNA/DNA and other nucleic acid substrates, as well as its ability to dissociate DNA-RNA hybrids and R-loops, including plasmid-based physiologically relevant R-loops. In Aim 3, we will purify ALYREF and CHTOP, and define their influence on UAP56’s activity in the unwinding of DNA- RNA hybrids and R-loop resolution in reconstituted biochemical systems and in cells. The results from our project will shed light on the general mechanism of co-transcriptional R-loop processing and genome preservation. We expect our endeavors to contribute toward the development of novel strategies to prevent pathogenic R-loops formation and to treat neurodegenerative disease and cancer caused by their accumulation. ! 1!