Investigating the Role of Transcription on DNA Damage at Early Replicating Fragile Sites

项目摘要/摘要 基因组不稳定是癌症的一个标志。这种不稳定性始于DNA损伤，即 不忠实地修复了。复制应激是这些损伤和转录的主要来源 极大地增强了复制压力。早期复制的脆弱部位(ERF)是特定的位置 在S早期经历反复DNA损伤的基因组中 复制压力。当ERF被映射到小鼠的原代B细胞时，发现它们 在基因丰富的区域被发现，并在很大程度上与重复的扩增和 弥漫性大B细胞淋巴瘤中的缺失。目前尚不清楚为什么这些部位容易携带DNA。 在ERF或癌症中的损害。在一个位点上显示转录的减少 降低了染色体的脆性。此前已有研究表明，两种物质的碰撞 复制叉(RF)和RNA聚合酶(RNAP)是诱变的。转录的 基因组早期复制区域的失调可能在该染色体中起作用。 脆弱。假设是早期复制区域转录活性的增加 由于射频的空间和时间共定位，将导致DNA损伤水平升高 和RNAP。这些实验将在原代小鼠B细胞中进行，因为它们独特的 转录图谱和正常的核型将使我们能够准确地记录与 野生型。第一个目的是检验这一假说，目的是在早期调节转录 通过靶向与任一者连接的催化缺陷CRISPR-dCas9来复制区域 转录激活因子或抑制因子。这将允许在特定情况下对转录进行调节 精神错乱。DNA损伤将通过观察染色体异常来测量 用荧光原位杂交技术(FISH)检测基因座。这种方法将展示如何在 转录活性影响早期复制区的DNA损伤水平。这个 第二个目标，检验这一假设，将在基因组中找到既有RF又有RF的位置 RNAP共定位于S早期。以在空间和时间上同时定位RF和RNAP 定位后，我将对RNAP和新生DNA进行顺序免疫沉淀(IP)， 用5-乙炔基-2‘-脱氧尿苷标记这些位置的DNA将被测序并 DNA损伤将使用FISH进行量化。连续的IP实验将为我们提供 在S早期具有RF和RNAP共定位的基因组基因座。这些数据加在一起将 提示转录编程的变化导致了RF和RF之间的冲突 在癌症中观察到RNAP会产生反复的DNA损伤。

Project Summary/Abstract Genome instability is a hallmark of cancer. This instability initiates with a DNA lesion that is unfaithfully repaired. Replication stress is a major source of these lesions and transcription greatly enhances replication stress. Early replicating fragile sites (ERFS) are specific locations in the genome that experience recurrent DNA damage in early S-phase in the presence of replication stress. When ERFS were mapped in mouse primary B-cells, it was found that they are found in in gene rich areas and largely overlap with recurrent sites of amplifications and deletions in diffuse large B-cell lymphoma. It is not known why these sites are prone to DNA damage at ERFS or in the cancer. It was shown at one site that a decrease in transcription decreased chromosome fragility. It has been previously shown that collisions between replication forks (RF) and RNA polymerase (RNAP) are mutagenic. Transcriptional dysregulation in early replicating regions of the genome may play a role in this chromosome fragility. The hypothesis is that an increase in transcriptional activity in early replicating regions will lead to elevated levels of DNA damage due to the spatial and temporal co-localization of RF and RNAP. These experiments will be performed in primary mouse B-cells, as their distinct transcriptional profile and normal karyotype will allow us to accurately document deviations from wild type. The first aim, to test the hypothesis, will be modulating transcription at early replicating regions by targeting a catalytically defective CRISPR-dCas9 tethered to either transcriptional activators or repressors. This will allow the modulation of transcription at specific loci. The DNA damage will be measured by observing chromosomal abnormalities at the same loci using fluorescence in situ hybridization (FISH). This approach will show how alterations in transcriptional activity influence the levels of DNA damage in early replicating regions. The second aim, to test the hypothesis, will be finding sites in the genome that have both RF and RNAP co-localized in early S-phase. To locate RF and RNAP spatially and temporally co- localizing, I will perform a sequential immunoprecipitation (IP) for RNAP and nascent DNA, labeled with 5-ethynyl-2'-deoxyuridine The DNA at these locations will be sequenced and the DNA damage will be quantified using FISH. The sequential IP experiment will provide us with genomic loci that have both RF and RNAP co-localized in early S-phase. Together the data will suggest that changes in transcriptional programming leads to conflicts between RF and RNAP generating recurrent DNA damage observed in cancer.