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

人类 UAP56 解旋酶在共转录 R 环解析和基因组维护中的作用

• 批准号: 10046203
• 负责人: XIAOYU XUE
• 金额: $ 43.64万
• 依托单位: TEXAS STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10046203
• 关键词: ATP phosphohydrolase; Binding; Biochemical; Cancer Etiology; Cells; Comet Assay; Complex; Conflict (Psychology); Cytosine; DNA; DNA Damage; DNA Double Strand Break; Data; Defect; Development; Dissociation; Excision; Gamma-H2AX; Genes; Genetic Transcription; Genome; Genome Stability; Genomic Instability; Genomics; Goals; Hela Cells; Human; Hybrids; Hydrolysis; Immunofluorescence Immunologic; Immunoglobulin Class Switching; Immunoglobulin Switch Recombination; Impairment; In Vitro; Lead; Light; Maintenance; Malignant Neoplasms; Maps; Mediating; Methods; Mission; Modeling; Molecular; Monitor; Monoclonal Antibodies; Motor; National Institute of General Medical Sciences; Neurodegenerative Disorders; Nucleic Acid Binding; Nucleic Acids; Pathogenicity; Pathologic; Physiological; Plasmids; Predisposition; Prevention; Process; Proteins; RNA; RNA Helicase; RNA Polymerase II; RNA Processing; RNA-Binding Proteins; Regulation; Resolution; Role; Series; Small Interfering RNA; Source; Specificity; Stress; Structure; System; Testing; Work; base; bisulfite sequencing; cofactor; helicase; insight; mutant; novel; novel strategies; overexpression; preservation; prevent; reconstitution; replication stress; transcription factor; transcription termination

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!

项目概要 共转录 R 环是长 DNA-RNA 杂合体，可在 RNA 聚合酶 II 转录过程中产生。 虽然 R 环具有重要的生理功能，例如类别转换重组和促进 转录终止、持久性 R 环是 DNA 损伤、复制应激和 基因组不稳定。 R 环分辨率缺陷见于神经退行性疾病和癌症。细胞有 进化出不同的策略来防止病理性 R 环的积累。其中一种机制依赖于 人类中保守的 THO 复合物及其相关的共转录因子 UAP56 解旋酶。然而， UAP56在预防R环中发挥作用的详细机制仍有待确定。 我们的初步数据表明，UAP56 的耗尽会导致 R 环积累和 R 环介导 基因组不稳定。此外，纯化的 UAP56 比 RNA-RNA 更擅长解旋 RNA-DNA 杂交体 复式。它还可以在体外解离模型 R 环。基于这些发现，我们假设 UAP56 参与 并解离共转录 R 环结构以防止其在细胞中积累。我们还假设 UAP56的R环解离活性在转录过程中需要通过其结合来严格调节 合作伙伴 ALYREF 和 CHTOP，两种 RNA 结合蛋白。该 R15 提案的总体目标是确定 UAP56 如何帮助消除有害的共转录 R 环，以及该活性如何受其辅助因子调节 以维持基因组稳定性。在这个项目中，我们将通过三个具体目标来实现我们的目标：（1）研究共同 UAP56在细胞中的转录R环去除和基因组维持功能； (2)确定R环 UAP56体外解析功能； (3)表征UAP56在R环去除中的调节作用 生理搭档，ALYREF 和 CHTOP。在目标 1 中，我们将检查 R 环水平和 DNA 损伤 使用一系列细胞和生化方法（包括使用免疫荧光 (IF)）去除 UAP56 细胞 S9.6 单克隆抗体，一组用于 DNA-RNA 杂合积累的靶基因中的 DRIP-qPCR，亚硫酸氢盐 对目标基因进行测序，监测胞嘧啶的转化，DRIPc-seq 在基因组规模上绘制 R 环图， 彗星测定和γH2AX焦点形成。在目标 2 中，利用高度纯化的 UAP56 及其解旋酶死亡突变体，我们 将研究其与一系列RNA/DNA和其他核酸底物的关联，以及其能力 解离 DNA-RNA 杂合体和 R 环，包括基于质粒的生理相关 R 环。瞄准 3、我们将纯化ALYREF和CHTOP，并确定它们对UAP56在DNA解旋中的活性的影响- 重组生化系统和细胞中的 RNA 杂合体和 R 环解析。我们项目的结果 将揭示共转录 R 环加工和基因组保存的一般机制。我们 期望我们的努力能够为预防致病性 R 环的新策略的开发做出贡献 形成并治疗由其积累引起的神经退行性疾病和癌症。 ！ 1！

项目成果

DDX47, MeCP2, and other functionally heterogeneous factors protect cells from harmful R loops.

• DOI: 10.1016/j.celrep.2023.112148
• 发表时间: 2023-03-28
• 期刊: Cell reports
• 影响因子: 8.8

Multifaceted regulation of the sumoylation of the Sgs1 DNA helicase.

• DOI: 10.1016/j.jbc.2022.102092
• 发表时间: 2022-07
• 期刊: JOURNAL OF BIOLOGICAL CHEMISTRY
• 影响因子: 4.8
• 作者: Li, Shibai;Mutchler, Ashley;Zhu, Xinji;So, Stephen;Epps, John;Guan, Danying;Zhao, Xiaolan;Xue, Xiaoyu
• 通讯作者: Xue, Xiaoyu