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。 当面临DNA损伤或染色体中的其他障碍时，分叉会停滞，从而产生复制压力。 这种压力通常会诱导DNA双链断裂，当不适当地诱导DNA双链断裂时， 修复.为了科普这种对基因组完整性的侮辱，细胞已经进化出了解决停滞分叉的机制 修复双链断裂这两种机制都受到ATR激酶的调节。特别是ATR 对于脆性位点的稳定性是重要的，脆性位点是易于断裂的特定基因组区域， 当复制受到干扰时会发生突变。最近的证据表明，复制应力和双链 断裂可以由R环诱导，R环是一种由RNA：DNA杂交体组成的三链结构， 单链DNA当RNA：DNA杂交体稳定存在时，在转录过程中某些基因产生R环。 形成了当发生在正常水平以上时，R环可能导致转录相关的诱变， 干扰DNA复制，并导致脆性位点断裂。考虑到R环可能会导致 复制应力和双链断裂在脆性网站，ATR保护脆性网站从断裂，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环积累时，ATR被激活。基于这一发现，我的目标1 将探索R环形成如何触发ATR激活。我的初步数据还表明， 具有升高的R-环水平的细胞增加了DNA断裂，这表明ATR保护基因组免受R-环的影响， 导致DNA损伤。我将研究ATR如何在Aim 2中扮演这个角色。这两个目标的实验 可能揭示和阐明R环DNA损伤反应中关键组分的功能。此外，本发明的目的是， 我发现R环形成序列在早期复制脆弱位点的基因中富集。其中一 IRF 4基因经常参与淋巴瘤中的致癌易位，并被预测形成一个非常重要的 长R环在我的目标3中，我将诱导IRF 4在其染色体位点的表达，测试它是否产生 R环，并确定ATR是否保护其免受R环诱导的脆性。这些实验可能揭示了 转录和随后的R-环形成对于在特定染色体位点引起脆性的重要性。 考虑到癌症中许多早期复制脆弱位点的基因发生突变，我的研究可能有助于将R环- 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 拟议的研究可能会大大扩展我们在这一新兴领域的知识，并为其他研究提供基础。 研究探索R环在肿瘤发生中的可能作用。

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.