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

摘要 基因组的忠实复制对生存至关重要。在S期，DNA合成发生在复制叉处。 当面临DNA损伤或其他染色体障碍时，叉子就会停滞不前，造成复制压力。 这种压力通常会导致dna双链断裂，如果处理不当，可能会导致基因组的不稳定。 修好了。为了应对这种对基因组完整性的侮辱，细胞进化出了解决停滞不前的分叉的机制 修复双链断裂。这两种机制都受ATR激酶的调节。特别是，ATR 对于脆弱部位的稳定性很重要，这些脆弱部位是容易断裂的特定基因组区域 当复制受到干扰时的突变。最近的证据表明，复制应激和双链 断裂可以由R-环诱导，R-环是一种由RNA：DNA杂交和移位组成的三链结构 单链DNA。当RNA：DNA杂交体稳定时，在转录过程中某些基因会产生R环 形成了。当发生在正常水平以上时，R-环可能会导致转录相关突变， 干扰DNA复制，并导致脆弱部位的断裂。鉴于R-环可能会导致这两个 复制压力和脆弱部位的双链断裂，ATR保护脆弱部位不受破坏，即 假设ATR是R-环的重要感受器，并抑制R-环相关的基因组不稳定性。 我的初步数据显示，当R环累积时，ATR被激活。基于这一发现，我的目标是1 将探索R环的形成如何触发ATR激活。我的初步数据还显示，ATR抑制在 R-loop水平升高的细胞会增加DNA断裂，这表明ATR保护基因组免受R-loop- 诱导DNA损伤。我将调查ATR如何在Aim 2中扮演这一角色。 可能揭示和阐明R-环引起的DNA损伤反应中的一个关键成分的功能。另外， 我发现R-Loop形成序列在早期复制的脆性部位富含基因。这其中的一个 IRF4基因经常参与淋巴瘤的致癌易位，并被预测形成一种非常 长长的R环。在我的目标3中，我将在其染色体上诱导性地打开IRF4的表达，测试它是否产生 R-环，并确定ATR是否保护它免受R-环诱导的脆弱性。这些实验可能揭示出 转录和随后的R-环的形成对在特定染色体座位上引发脆性的重要性。 鉴于癌症早期复制脆弱部位的许多基因都发生了突变，我的研究可能有助于将R-loop-loop- 诱导不稳定性导致肿瘤发生，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一新兴领域的知识，并为其他 探索R-环在肿瘤发生中的可能作用的研究。

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.