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。从基因组非编码区编码的多个位点（复制起点）开始，整个基因组的复制完成是高度调控的，对任何物种的生存都至关重要。尽管这一过程具有非常基本的性质，但迄今为止，在后生动物细胞中只有不到几十个复制起源被表征。我们对基因组中起源数量、复制终止位点和复制子排列的了解仍然非常有限。在这里，我们建议开发一种基于Okazaki片段链分析的通用基因组尺度定位方法，该方法使用由Us /ymes型生成的短序列特异性标签。利用出芽酵母作为可处理的模型系统，对该方法的详细步骤进行验证和优化。通过这种方法，不仅可以以高分辨率并行绘制复制起点，还可以绘制出定义基因组中每个复制子所需的叉终止位点。到目前为止，还不可能大规模地直接绘制分叉终止的地点。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，该应用程序的后半部分将重点放在开发有效的方案上，以便在基因组的选定区域分离冈崎片段，并去除大部分重复序列。这种降低的复杂性是实现有限长度的标签与目标序列的唯一匹配所必需的。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，人类基因组的大规模绘制，以及其他后生动物的基因组，可以充满信心地开始。在这些综合数据集的许多可能的用途中，对各种后生动物基因组的比较检查将最肯定地揭示染色体背景下复制子的组织和保存，进一步阐明嵌入在基因组非编码区域的最重要功能信号之一的进化。很有可能，当已知起源数量增加时，共识序列基序或特征也可能被发现，它们在定义和调节复制起源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.