Investigating the Novel Function of ATR Checkpoint Kinase in R-Loop Responses

研究 ATR 检查点激酶在 R 环反应中的新功能

基本信息

批准号：
9188696
负责人：
Dominick Amaral Matos
金额：
$ 3.11万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-01 至 2018-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9188696
关键词：
Address Antibodies B-Lymphocytes Binding Cells Chromatin Chromosome Fragile Sites Chromosomes Clustered Regularly Interspaced Short Palindromic Repeats DNA DNA Damage DNA Double Strand Break DNA biosynthesis DNA replication fork Data Double Strand Break Repair Foundations Genes Genetic Transcription Genome Genomic Instability Genomic Segment Hela Cells Hybrids IRF4 gene Knowledge Lead Link Lymphoma Malignant Neoplasms Metaphase Spread Mutagenesis Mutate Oncogenic Phosphotransferases Play Process Pulsed-Field Gel Electrophoresis RNA Recruitment Activity Role S Phase Single-Stranded DNA Site Source Stress Structure System Testing Transcriptional Activation Western Blotting abstracting base cancer therapy chromosome replication coping genome integrity inhibitor/antagonist knock-down novel overexpression prevent repaired research study response sensor tumorigenesis

项目摘要

Abstract Faithful replication of the genome is vital for survival. During S phase, DNA synthesis occurs at replication forks. When facing DNA damage or other impediments in chromosomes, forks are stalled creating replication stress. This stress often induces DNA double-strand breaks, which can lead to genomic instability when improperly repaired. To cope with this insult to genomic integrity, cells have evolved mechanisms to resolve stalled forks and repair double-strand breaks. Both of these mechanisms are regulated by the ATR kinase. In particular, ATR is important for the stability of fragile sites, which are specific genomic regions prone to breakage and mutagenesis when replication is perturbed. Recent evidence suggested that replication stress and double-strand breaks can be induced by R-loops, a three-stranded structure consisting of a RNA:DNA hybrid and displaced single-stranded DNA. R-loops arise from certain genes during transcription when RNA:DNA hybrids are stably formed. When occurring above normal levels, R-loops may cause transcription-associated mutagenesis, interfere with DNA replication, and lead to breakage at fragile sites. Given that R-loops may give rise to both replication stress and double-strand breaks at fragile sites, and that ATR protects fragile sites from breakage, I hypothesis that ATR is an important sensor of R-loops, and suppresses R-loop-associated genomic instability. My preliminary data suggest that ATR is activated when R-loops accumulate. Based on this finding, my Aim 1 will explore how R-loop formation triggers ATR activation. My preliminary data also show that ATR inhibition in cells with elevated R-loop levels increases DNA breaks, suggesting that ATR protects the genome from R-loop- induced DNA damage. I will investigate how ATR performs this role in Aim 2. The experiments in these two aims may reveal and elucidate the function of a key component in the DNA damage response to R-loops. Additionally, I found that R-Loop Forming Sequences are enriched in genes at early replicating fragile sites. One of these genes, IRF4, is frequently involved in oncogenic translocations in lymphomas and is predicated to form a very long R-loop. In my Aim 3, I will inducibly turn on IRF4 expression at its chromosomal locus, test if it generates R-loops, and determine if ATR protects it from R-loop-induced fragility. These experiments may reveal the importance of transcription and subsequent R-loop formation to instigating fragility at specific chromosomal loci. Given that many genes at early replicating fragile sites are mutated in cancer, my studies may help link R-loop- induced instability to tumorigenesis, which could provide new opportunities for cancer therapy. Overall, my proposed studies may greatly expand our knowledge in this emerging field and provide a foundation for other studies exploring the possible role of R-loops in tumorigenesis.

抽象的基因组的忠实复制对于生存至关重要。在 S 期，DNA 合成发生在复制叉处。当面临 DNA 损伤或染色体中的其他障碍时，分叉就会停滞，从而产生复制压力。这种压力通常会导致 DNA 双链断裂，如果处理不当，可能会导致基因组不稳定。修复了。为了应对这种对基因组完整性的损害，细胞进化出了解决停滞叉的机制并修复双链断裂。这两种机制均受 ATR 激酶调节。特别是，ATR 对于脆弱位点的稳定性很重要，脆弱位点是容易断裂的特定基因组区域当复制受到干扰时发生突变。最近的证据表明复制压力和双链 R 环可以诱导断裂，R 环是一种由 RNA:DNA 杂合体和置换组成的三链结构单链DNA。当 RNA:DNA 杂交体稳定存在时，R 环在转录过程中由某些基因产生形成。当 R 环出现高于正常水平时，可能会导致转录相关突变，干扰 DNA 复制，并导致脆弱位点断裂。鉴于 R 环可能会产生以下两种情况脆弱位点的复制压力和双链断裂，并且 ATR 可以保护脆弱位点免于断裂，我假设 ATR 是 R 环的重要传感器，并抑制 R 环相关的基因组不稳定性。我的初步数据表明，当 R 环累积时，ATR 就会被激活。基于这一发现，我的目标 1 将探讨 R 环形成如何触发 ATR 激活。我的初步数据还表明，ATR 抑制 R 环水平升高的细胞会增加 DNA 断裂，表明 ATR 可以保护基因组免受 R 环的影响诱导DNA损伤。我将研究ATR如何在目标2中发挥这个作用。这两个目标中的实验可能揭示并阐明 R 环 DNA 损伤反应中关键成分的功能。此外，我发现 R 环形成序列富含早期复制脆弱位点的基因。其中之一基因 IRF4 经常参与淋巴瘤的致癌易位，并且预计会形成一个非常长R环。在我的目标 3 中，我将在其染色体位点诱导开启 IRF4 表达，测试它是否生成 R 环，并确定 ATR 是否可以保护其免受 R 环引起的脆弱性。这些实验可能揭示转录和随后的 R 环形成对于引发特定染色体位点脆弱性的重要性。鉴于早期复制脆弱位点的许多基因在癌症中发生突变，我的研究可能有助于将 R 环与诱导肿瘤发生的不稳定性，这可能为癌症治疗提供新的机会。总的来说，我的拟议的研究可能会极大地扩展我们在这个新兴领域的知识，并为其他领域提供基础探索 R 环在肿瘤发生中可能作用的研究。