Genomic Mapping of Replication Origins in Higher Eukaryotes by Okazaki Analysis

描述（由申请人提供）：DNA复制是最基本的生物过程之一。从主要在基因组的非编码区编码的多个位点（复制起点）开始，整个基因组的复制的完成是高度调节的，并且对于任何物种的生存都是必不可少的。尽管这一过程非常基本，但迄今为止，后生动物细胞中的复制起点的特征还不到几十个。我们对基因组中复制起点的数目、复制终止的位点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，基因组规模的映射方法的基础上使用短的，序列特异性标签产生的lype Us en/ymes的冈崎片段的链分析。以芽殖酵母为模型系统，对该方法的具体步骤进行了验证和优化。通过这种方法，不仅复制起点被平行地映射到高分辨率，而且定义基因组中每个单独复制子所需的叉终止位点也被映射到高分辨率。到目前为止，还不可能直接大规模地绘制分叉终止的地点。为了将该方法应用于含有相当大量重复序列的更大的人类基因组，本申请的后半部分集中于开发有效的方案以分离去除了大部分重复序列的基因组的选择区域上的冈崎片段。需要这种降低的复杂性来实现有限长度的标签与靶序列的独特匹配。为此，将使用ENCODE项目选择的部分序列。当这些目标实现时，这种方法的可行性将得到充分证明，并且可以有信心地开始大规模绘制人类基因组以及其他后生动物基因组。在许多可能的效用，这种全面的数据集是比较检查的各种后生动物基因组，这将最肯定地揭示了组织和保存的复制子在染色体的情况下，进一步阐明了一个最重要的功能信号嵌入在非编码区的基因组的进化。当已知的复制起点数量增加时，可能也会发现共有序列基序或签名，它们在定义和调节复制起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.