Genomic Mapping of Replication Origins in Higher Eukaryotes by Okazaki Analysis

描述(申请人提供)：DNA复制是最基本的生物学过程之一。从主要在基因组非编码区编码的多个位点(复制起点)开始，完成整个基因组的复制是高度调控的，对任何物种的生存都是必不可少的。尽管这一过程具有非常基本的性质，但到目前为止，后生动物细胞中只有不到几十个复制起点被表征。我们对基因组中起始点的数量、复制终止的位置和复制子的排列的了解仍然非常有限。在这里，我们建议开发一种通用的基因组规模的作图方法，该方法基于对Okazaki片段的链分析，使用lype us en/ymes生成的短序列特定标签。以发芽酵母为模型系统，对该方法的具体步骤进行验证和优化。利用这种方法，不仅复制起点被并行地映射到高分辨率，而且定义基因组中每个单独的复制子所需的分叉终止点也被映射到高分辨率。到目前为止，还不可能直接在大范围内绘制叉子终止地点的地图。为了将这种方法应用于包含大量重复序列的更大的人类基因组，本申请的后半部分专注于开发有效的方案，以分离基因组选定区域上的Okazaki片段，并去除大部分重复序列。需要这种降低的复杂性来实现有限长度的标签与目标序列的唯一匹配。为此，将使用ENCODE项目选择的序列的一部分。当这些目标实现时，这种方法的可行性将得到充分证明，人类基因组以及其他后生动物基因组的大规模绘制工作就可以满怀信心地开始了。在这种综合数据集的许多可能的用途中，包括对各种后生动物基因组的比较研究，这将最肯定地揭示复制子在染色体范围内的组织和保守，进一步阐明嵌入基因组非编码区的最重要的功能信号之一的进化。当已知起始点的数量增加时，可能还会发现一致的序列基序或签名，这可能在定义和调节复制起始点的功能方面发挥关键作用。

DESCRIPTION (provided by applicant): DNA replication is one of the most fundamental biological processes. Starting from multiple sites (replication origins) encoded mostly in non-coding regions of the genome, the completion of duplication of the entire genome is highly regulated and essential for the survival of any species. Despite the very basic nature of this process, less than a few dozen of replication origins in metazoan cells have been characterized to date. Our knowledge on the number of origins, the sites of replication termination and the arrangement of replicons in the genome remains very limited. Here, we propose to develop a versatile, genomic scale mapping method based on strand analysis of Okazaki fragments using short, sequence specific tags generated by lype Us en/ymes. Using the budding yeast as a tractable model system, the detailed procedures of this approach will be validated and optimized. With this method, not only are the replication origins mapped to a high resolution in parallel, but also the fork termination sites that are required to define each individual replicons in the genome. To date, it has not been possible to map the sites of fork termination directly on a large scale. To apply this approach to the much larger human genome containing considerable amount of repeat sequences, the later half of this application is focused on the development of effective protocols to isolate Okazaki fragments on a select region of the genome with the majority of repeat sequences removed. This reduced complexity is required to achieve unique matching of tags of limited length to target sequences. For this purpose, a portion of the sequences selected by the ENCODE project will be used. When these objectives are accomplished, the feasibility of this approach will have been fully demonstrated, and large scale mapping of the human genome, as well as other metazoan genomes, can be initiated with confidence. Among many possible utilities of such comprehensive datasets is the comparative examination of various metazoan genomes that will most certainly reveal the organization and conservation of replicons in the chromosomal context, further elucidating the evolution of one of the most important functional signals embedded in noncoding regions of the genome. It is probable that when the number of known origins increases, consensus sequence motifs or signatures may also be discovered, which can play critical roles in defining and regulating the function of replication origins.