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首页> 外文学位 >General Replicators and Replication Origins in Human Cells And The Role of Yeast ORC in Mediating Histone Methylation.
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General Replicators and Replication Origins in Human Cells And The Role of Yeast ORC in Mediating Histone Methylation.

机译：人体细胞中的一般复制子和复制起点，以及酵母ORC在介导组蛋白甲基化中的作用。

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摘要

Initiation of DNA replication is controlled by the cis-acting replicators and the trans-acting initiator and initiation proteins that interact with replicators to initiate replication at replication origins. The replicators and origins have been defined as specific DNA sequences in yeasts. However, although over one hundred unique metazoan replicators/origins have been identified, searching for general metazoan replicators is less fruitful. Here, I report a group of general human replicators identified by chromatin immunoprecipitation (ChIP) using antibodies against ORC and MCM proteins followed by unbiased cloning of the precipitated DNA. The interactions of the putative replicators with multiple initiation proteins were confirmed by ChIP assays with multiple PCR primer pairs to detect the precipitated DNA and a more stringent re-ChIP assay. The putative replicators are part of LINE1 (L1; long interspersed nuclear elements), and are homologous to one another and also to tens of thousand other loci distributed relatively evenly in the human genome, with a density of one in every ∼100 kb. The DNA sequence, per se, shares some features of replication origins. It was also demonstrated that the replication origins were located near the replicators using nascent strand abundance assays with either alkaline purified or BrdU labeled nascent DNA. In addition, the DNA combing assay confirmed that the in vivo DNA replication was initiated in the area of the putative replicators. Furthermore, episomal replication assays showed that the replicator/origin-containing plasmids, but not the control plasmids, became Dpn1-resisitant after transfection into human cells, indicating duplication of the replicator/origin-containing plasmids. This study identified a group of human replicators and replication origins that are part of L1, and they may represent the general replicators and replication origins responsible for the duplication of the bulk of the human genome.;The hetero-hexameric origin recognition complex (ORC) has been implicated in many cellular activities, including DNA replication, transcriptional control, heterochromatin assembly, centromere and telomere function, and so on. Here, I report a new function for ORC in mediating histone methylation. Using the yeast two-hybrid system I identify a physical interaction between Orc2p and Spp1p, a member of the Set1 complex, and I demonstrate the interaction between the endogenous ORC and Spp1p by co-immunoprecipitation (co-IP) from yeast extracts. Furthermore, I find that Orc2p physically interacts with trimethylated histone 3 lysine 4 (H3K4) on chromatin by co-IP. Finally, I show that the tri-methylation of H3K4 is decreased in orc2-1 cells and abolished in orc2-1 spp1Delta double mutants. These data reveal a novel facet of ORC in mediating histone methylation in collaboration with Spp1p, and demonstrate a connection between ORC and chromatin structure via the Set1 complex.
机译：DNA复制的起始受顺式作用复制子和反式启动子以及与复制子相互作用以在复制起点起始复制的起始蛋白控制。复制子和起源已被定义为酵母中的特定DNA序列。然而，尽管已经鉴定出一百多种独特的后生代复制子/起源，但是寻找通用的后生代复制子的成果较少。在这里，我报告了一组普通的人类复制子，这些复制子是通过使用针对ORC和MCM蛋白的抗体的染色质免疫沉淀（ChIP）鉴定的，然后无偏析地沉淀了DNA。推定复制子与多种起始蛋白的相互作用通过ChIP分析与多个PCR引物对进行检测，以检测沉淀的DNA，并进行更严格的re-ChIP分析。假定的复制子是LINE1（L1；长而散布的核元件）的一部分，彼此同源，并且与人类基因组中相对均匀分布的数万个其他基因座同源，密度约为每100 kb一个。 DNA序列本身具有复制起点的某些特征。还证明了使用碱性纯化或BrdU标记新生DNA的新生链丰度测定法，复制起点位于复制子附近。另外，DNA梳理测定证实了体内DNA复制在假定的复制子区域内开始。此外，附加型复制测定法表明，复制子/含起源的质粒，而不是对照质粒，在转染入人细胞后变为Dpn1-抗性，表明复制子/含起源的质粒重复。这项研究确定了一组属于L1的人类复制子和复制起点，它们可能代表负责复制人类基因组大部分的一般复制子和复制起点。;异六聚体起源识别复合体（ORC）已经涉及许多细胞活动，包括DNA复制，转录控制，异染色质装配，着丝粒和端粒功能等等。在这里，我报道了ORC在介导组蛋白甲基化中的新功能。使用酵母双杂交系统，我确定了Orc2p和Spp1p（Set1复合物的成员）之间的物理相互作用，并且我通过酵母提取物中的共免疫沉淀（co-IP）演示了内源性ORC和Spp1p之间的相互作用。此外，我发现Orc2p通过co-IP与染色质上的三甲基化组蛋白3赖氨酸4（H3K4）发生物理相互作用。最后，我证明在orc2-1细胞中，H3K4的三甲基化程度降低，而在orc2-1 spp1Delta双突变体中则消失。这些数据揭示了ORC与Spp1p协同介导组蛋白甲基化的一个新方面，并证明了SetC复合体在ORC和染色质结构之间具有联系。

著录项

  • 作者

    Kan, Junsuo.;

  • 作者单位

    Hong Kong University of Science and Technology (Hong Kong).;

  • 授予单位 Hong Kong University of Science and Technology (Hong Kong).;
  • 学科 Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 144 p.
  • 总页数 144
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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