Dissecting the impact of RNA on DNA replication and chromatin structure

项目摘要 DNA复制和转录机制之间的冲突（复制-转录冲突）出现 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 损伤，因此对基因组完整性的威胁，我们缺乏详细的分子理解， DNA上转录RNA聚合酶的存在影响复制的进程和机制 通过这些复制-转录冲突引起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rNTPs和dNTPs之间的不完全区分， 复制型DNA聚合酶，大量的核糖核苷酸被错误地掺入到基因组中， 每一轮复制;这种负担估计为在人细胞中每次细胞分裂> 1百万个核糖核苷酸， 使核糖核苷酸成为真核DNA中最丰富的损伤。错误掺入的核糖核苷酸是 通过核糖核苷酸切除修复（RER）途径去除，并且受损的去除与几种人类疾病有关。 疾病然而，核糖核苷酸如何影响染色质-DNA的高级结构- 或者相反染色质如何影响粗面内质网。此外，尚未确定是否所有核糖核苷酸 也同样易于修复，或者可能是基因组RER效率差异的基础。 拟议的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如何影响复制进程和基因组完整性 产生于转录的基因。为了实现这一点，我们使用最近开发的定量方法来测定 高分辨率的复制体全基因组移动，结合全基因组询问， DNA双链断裂的形成和一种新的测定法来定位在被抑制的背景下的新生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的组合来描述如何 核糖核苷酸使核小体（染色质的基本重复单位）不稳定，核小体如何影响RER 启动的RNase H2酶，并阐明在基因组中的所有位点的RER的动力学。 负责DNA复制、转录和DNA修复的机制在整个过程中是高度保守的。 真核生物这两个项目都将在芽殖酵母酿酒酵母中进行：小基因组， 快速复制和遗传操作性。酿酒酵母使这一理想的模型，其中研究 基本生物过程的交叉点。因此，这项工作的结果将提供分子 对人类基因组不稳定性的深入了解，将直接适用于我们对病因学的理解 和癌症的进展以及罕见疾病，包括Aicardi-Goutières综合征。

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.