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Mapping Origins of DNA Replication in the Genome

绘制基因组中 DNA 复制的起源图

基本信息

批准号：
9331710
负责人：
SUSAN A GERBI
金额：
$ 32.5万
依托单位：
BROWN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-15 至 2019-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9331710
关键词：
Antibodies Applications Grants Basal Cell Nevus Syndrome Binding Binding Sites Biological Buffers Cancer cell line Cell Cycle Cell Line Cells Comb animal structure Coupled DNA DNA biosynthesis DNA replication origin Data Data Set Development Digestion Emetine Eukaryota Exonuclease Fluorescent in Situ Hybridization Frequencies G-Quartets Genome Genomic Instability Genomic approach Hela Cells Human Human Cell Line Human Genome Individual Inherited Lead Life Link MCF7 cell Maps Methods Normal Cell Parents Positioning Attribute Preparation Process Protocols documentation RNA primers Replication Initiation Replication Origin Resistance Ribonucleases Saccharomycetales Structure Sum Validation Wolf-Hirschhorn Syndrome base cancer cell cell type chromatin immunoprecipitation daughter cell experimental study genetic information genome-wide human disease internal control public health relevance yeast genome

项目摘要

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.

描述（由申请人提供）：我们建议鉴定人类基因组中定义DNA复制的所有潜在起点（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。其他人在绘制ORI全基因组图谱的prio结果中的分歧可能反映了通过λ核酸外切酶（Lexo）富集的新生链制备物中的污染。我们的初步结果表明，Lexo不能消化G四链体（G4）结构，因此，G4将显着污染新生链制剂。我们将通过标准化到匹配的内部控制和使用缓冲液条件来最大限度地减少Lexo偏差来纠正这个问题。这些校正将用于新生链测序（NS-seq）以在全基因组范围内定位活性ORI。此外，鉴定用于前导链合成的链开关将确认双向复制的ORI的位置。我们将首先将我们修改后的NS-seq方案应用于芽殖酵母，其中所有ORI在基因组中都是已知的，从而验证我们修改后的NS-seq方法。接下来，我们将修改后的NS-seq方案应用于三种人类细胞系的基因组，使我们能够比较正常细胞（GM 06990）和癌细胞（HeLa和MCF 7）中使用的活性ORI。我们将验证我们的结果，通过使用正交的方法，从我们的数据集的一些选定的ORI的DNA梳理。DNA梳理还将使我们能够确定单个DNA分子上活性ORI之间的间隔。染色质免疫沉淀（ChIP）将用于鉴定基因组中的所有潜在ORI（结合ORC 2的ORI）和活性ORI（结合Cdc 45的ORI）子集。为此，我们将采用ChIP-reChIP方法与ChIP-exo相结合。活性ORI处的Cdc 45结合位点也将用于验证通过NS-seq映射的活性ORI。我们实验的意义是双重的。首先，在生物学水平上，我们的研究结果的总和将允许我们对基因组中与所有潜在ORI的定义相关的特征进行分类，与这些特征的子集相关的特征成为活跃的ORI。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开发修订的协议，从而影响复制领域。