Mapping Origins of DNA Replication in the Genome

描述(申请人提供)：我们建议确定人类基因组中定义DNA复制的所有潜在起始点(ORI)的特征，以及定义所有潜在ORI的哪个子集将被激活以启动DNA复制的特征。其他人在PRIO结果中对Oris全基因组图谱的不一致可能反映了富含lambda核酸外切酶(LEXO)的新生链制剂中的污染。我们的初步结果显示，Lexo不能消化G四链(G4)结构；因此，G4将严重污染新生的链制剂。我们将通过归一化到匹配的内部控制和使用最大限度地减少词汇偏差的缓冲条件来纠正这个问题。这些校正将被用于新生的链测序(NS-SEQ)，以定位整个基因组范围内活跃的Oris。此外，识别用于先导链合成的链开关将确定双向复制的ORI的位置。我们将首先将我们修订的NS-SEQ方案应用于所有ORI都在基因组中已知的萌芽酵母，从而验证我们修订的NS-SEQ方法。接下来，我们将把修订后的NS-SEQ方案应用于三个人类细胞系的基因组，使我们能够比较正常细胞(GM06990)和癌细胞(HeLa和MCF7)使用的活性ORI。我们将使用从我们的数据集中选择的几个ORI的DNA正交法来验证我们的结果。DNA梳理还将使我们能够确定单个DNA分子上活跃的Ori之间的间距。染色质免疫沉淀(CHIP)将被用来识别基因组中所有潜在的ORI(那些结合ORC2的)和活性ORI的子集(那些结合CDC45的)。为此，我们将采用CHIP-reChIP和CHIP-EXO相结合的方法。活跃的ORIS上的CDC45结合位点也将用于验证NS-SEQ所映射的活跃的ORIS。我们实验的意义是双重的。首先，在生物学水平上，我们的结果的总和将允许对基因组中与所有潜在Oris和ORI的定义相关的特征进行关联 与这些成为活动ORI的子集相关的功能。其次，在技术层面，我们对Lexo偏见的纠正将使我们能够为NS-SEQ制定修订的协议，从而影响复制领域。

DESCRIPTION (provided by applicant): We propose to identify features in the human genome that define all potential origins (ORIs) of DNA replication and features that define which subset of all potential ORIs will be activated for initiation of DNA replication. The disagreement in prio results by others to map ORIs genome-wide may reflect contamination in nascent strand preparations that were enriched by lambda exonuclease (Lexo). Our preliminary results reveal that Lexo cannot digest G quadruplex (G4) structures; therefore, G4 will significantly contaminate nascent strand preparations. We will correct for this problem by normalization to a matched internal control and by use of buffer conditions that minimize Lexo biases. These corrections will be used in nascent strand sequencing (NS-seq) to map active ORIs genome-wide. Moreover, identification of the strand switch for leading strand synthesis will confirm the position of ORIs of bi-directional replication. We will first apply our revised NS-seq protocol to budding yeast where all ORIs are known in the genome, thus validating our revised NS-seq approach. Next, we will apply the revised NS-seq protocol to the genomes of three human cells lines, allowing us to compare active ORIs used by normal cells (GM06990) to those used in cancer cells (HeLa and MCF7). We will validate our results by use of the orthogonal approach of DNA combing for a few selected ORIs from our data set. DNA combing will also allow us to determine the spacing between active ORIs on single DNA molecules. Chromatin immunoprecipitation (ChIP) will be used to identify all potential ORIs in the genome (those that bind ORC2) and the subset that are active ORIs (those that bind Cdc45). For this, we will employ ChIP-reChIP approaches coupled with ChIP-exo. The Cdc45 binding sites at active ORIs will also serve to validate the active ORIs mapped by NS-seq. The significance of our experiments is two-fold. First, at the biological level, the sum of our results will allow correlatons to be drawn for features in the genome that correlate with the definition of all potential ORIs and the features that correlate with the subset of these that become active ORIs. Second, at the technical level, our corrections for Lexo biases will allow our development of revised protocols for NS-seq, thereby impacting the replication field.