Dissecting the impact of RNA on DNA replication and chromatin structure

项目总结 DNA复制和转录机制之间出现冲突(复制-转录冲突 在真核细胞中很常见。尽管这些冲突长期以来一直被研究为DNA的潜在来源 损害，因此，对基因组完整性的威胁，我们缺乏详细的分子理解 转录核糖核酸聚合酶的存在影响复制的进程及其机制(S) 这些复制-转录冲突会导致DNA损伤。 DNA复制过程中转录的另一个结果是核糖核苷三磷酸水平升高。 (RNTPS)--RNA聚合酶的底物。由于rntp和dntps之间的不完全区别， 在复制DNA聚合酶的过程中，大量的核苷酸被错误地结合到基因组中 每一轮复制；这一负担估计为人类细胞每个细胞分裂100万个核苷酸， 使核糖核苷酸成为目前真核DNA中含量最丰富的损伤。错误掺入的核糖核苷酸是 通过核糖核苷酸切除修复(RER)途径移除，受损移除与几个人类 疾病。然而，目前尚不清楚核苷酸如何影响染色质--DNA的高阶结构-- 或者相反，染色质是如何影响粗面内质网的。此外，还不能确定是否所有的核糖核苷酸 都同样易于修复，或者可能是通过基因组修复RER效率差异的原因。 拟议工作包括两个正在进行的项目： 第一个项目解决了方向依赖对复制进程和基因组完整性的影响 产生于转录后的基因。为了实现这一点，我们使用了一种最近开发的定量方法来分析 以高分辨率在全基因组范围内移动复制体，结合全基因组询问 DNA双链断裂的形成和一种新的方法在被捕者的背景下定位新的DNA链 复制分叉。 第二个项目使用全基因组分析和体外生物化学相结合的方法来描述 核糖核苷酸破坏核小体(染色质的基本重复单位)的稳定性，核小体如何影响粗面内质网 RNaseH2酶的启动，并阐明基因组中所有基因座上RER的动态。 负责DNA复制、转录和DNA修复的机器在整个过程中高度保守 真核生物。这两个项目都将在萌芽中的酿酒酵母中进行：小基因组， 酿酒酵母的快速复制和遗传可操作性使其成为研究酿酒酵母 基本生物过程的交集。因此，这项工作的结果将为分子 对人类基因组不稳定性的洞察，并将直接适用于我们对病因的理解 癌症和包括艾卡迪-古蒂埃综合征在内的罕见疾病的发展。

PROJECT SUMMARY Collisions between the DNA replication and transcription machineries (replication-transcription conflicts) appear to be common in eukaryotic cells. Although these conflicts have long been studied as a potential source of DNA damage and, therefore, a threat to genome integrity, we lack a detailed molecular understanding of how the presence of transcribing RNA polymerases on DNA affects the progress of replication, and of the mechanism(s) by which these replication-transcription conflicts give rise to DNA damage. Another consequence of transcription during DNA replication is elevated levels of ribonucleoside triphosphates (rNTPS) – the substrate for RNA polymerases. Due to incomplete discrimination between rNTPs and dNTPs by the replicative DNA polymerases, large numbers of ribonucleotides are mis-incorporated into the genome during each round of replication; this burden is estimated at > 1 million ribonucleotides per cell division in human cells, making ribonucleotides by far the most abundant lesion in eukaryotic DNA. Mis-incorporated ribonucleotides are removed via the ribonucleotide excision repair (RER) pathway, and impaired removal is linked to several human diseases. However, it is not known how ribonucleotides impact chromatin – the higher-order structure of DNA – or conversely how chromatin affects RER. Furthermore, it has not been determined whether all ribonucleotides are equally amenable to repair or what may underlie differences in RER efficiency through the genome. The proposed work encompasses two ongoing projects: The first project addresses how orientation-dependent effects on replication progression and genome integrity arise at transcribed genes. To achieve this, we use a recently developed quantitative method to assay the movement of the replisome genome-wide at high resolution, in combination with genome-wide interrogation of DNA double-strand break formation and a novel assay to map nascent DNA strands in the context of an arrested replication fork. The second project uses a combination of genome-wide assays and in vitro biochemistry to delineate how ribonucleotides destabilize nucleosomes (the basic repeating unit of chromatin), how nucleosomes affect RER initiation by the RNase H2 enzyme, and to elucidate the dynamics of RER at all loci in the genome. The machineries responsible for DNA replication, transcription, and DNA repair are highly conserved throughout eukaryotes. Both projects will be carried out in the budding yeast Saccharomyces cerevisiae: the small genome, rapid replication, and genetic manipulability of S. cerevisiae make this an ideal model in which to study the intersection of fundamental biological processes. Therefore, the results of this work will provide molecular insights into genome instability in humans, and will be directly applicable to our understanding of the etiology and progression of cancer as well as rare diseases including Aicardi-Goutières syndrome.