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Purification and characterisation of a novel dominant initiation factor for chromosomal DNA replication

染色体 DNA 复制的新型显性起始因子的纯化和表征

基本信息

批准号：
BB/K013378/1
负责人：
Torsten Krude
金额：
$ 47.13万
依托单位：
University of Cambridge
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2013
资助国家：
英国
起止时间：
2013 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FK013378%2F1
关键词：
Purification characterisation novel dominant initiation

项目摘要

Development and sustainability of a healthy multicellular organism depends on the accurate and well-controlled replication of its genome, in each cell each time before it divides. It is therefore of vital importance for human and animal health that underlying molecular mechanisms are in place, ensuring that DNA replication occurs only at the right time and place. The purpose of the proposed research is to characterise a novel molecular mechanism that regulates DNA replication during early vertebrate development.The regulation of DNA replication changes drastically during early development in vertebrate organisms. The best understood model organism for this regulation is the African clawed frog, Xenopus laevis. After a Xenopus egg is fertilised, it replicates its DNA rapidly during twelve short division cycles, and without any preference where to start. In fact, DNA replication starts already on individual chromosomes in mitosis. Extracts prepared from activated eggs recapitulate this regulation and replicate very rapidly any DNA that is added experimentally. After the initial twelve short division cycles, the embryo undergoes a transition (called the mid-blastula transition, or MBT) when DNA replication becomes more restrictively controlled. DNA replication is now separated from mitosis during the cell division cycle by distinct gap phases, and it initiates with a preference for particular chromosomal sites.The applicant's group has recently shown that a rather unusual factor becomes essential for the initiation of DNA replication at MBT in Xenopus and in zebrafish. This factor is not a protein, but a non-coding RNA termed Y RNA. Degradation of Y RNA has no effect on DNA replication before MBT at all, while in contrast, embryos without functional Y RNA spectacularly fail to replicate their DNA after MBT and die.The objective of this research project is to understand how DNA replication can initiate before MBT without requiring Y RNA function. Results from unpublished pilot experiments have established that there is a dominant initiation factor present in eggs and early embryos that overrides the requirement of Y RNAs for DNA replication in a dominant manner. The identity of this factor is unknown.The research proposed here is aimed at identifying and characterising this new and dominant initiation factor. In this project, we have three main objectives. (I) Purification. We will first purify this initiation factor from Xenopus egg extracts by standard chromatography techniques. We will monitor its activity in a reconstituted cell-free system for the initiation of DNA replication, which has been depleted of Y RNA function and thus depends on this novel factor. (II) Identification. Once purification has been achieved successfully, we will identify the dominant initiation factor by mass spectrometry. We will also identify the closest human relatives of this new factor. (III) Characterisation. The project will go two parallel ways here. On the one hand, we will characterise the normal biological function of this initiation factor in developing early Xenopus embryos. We will examine how much of this factor is present at key stages of embryo development. We will analyse how its activity is regulated at key stages, in particular around the MBT. On the other hand, we will also characterise the homologous protein in human cells. We will investigate whether or not it is present in a panel of human cells, including normal, senescent and cancer cells. We will express this factor in cells lacking this factor, and turn its expression off in those cells that express it. The aim is to investigate if this factor plays a role in normal human somatic cells, and to investigate the consequences for DNA replication and cell proliferation if this factor becomes activated at the wrong time and place.

健康的多细胞生物体的发展和可持续性取决于其基因组的准确和良好控制的复制，每次在每个细胞分裂之前。因此，对人类和动物健康至关重要的是，潜在的分子机制到位，确保DNA复制只发生在正确的时间和地点。本研究的目的是揭示脊椎动物早期发育过程中DNA复制调控的一种新的分子机制。对这种调节最了解的模式生物是非洲爪蟾（Xenopus laevis）。非洲爪蟾卵受精后，它在12个短暂的分裂周期中迅速复制DNA，并且没有任何偏好。事实上，DNA复制已经开始在有丝分裂的单个染色体上。从激活的卵制备的提取物重现了这种调节，并非常迅速地复制实验添加的任何DNA。在最初的12个短分裂周期之后，胚胎经历了一个过渡期（称为中期囊胚过渡期，或MBT），此时DNA复制受到更严格的控制。现在DNA复制与细胞分裂周期中的有丝分裂是分开的，由不同的间隙期，它开始与特定的染色体位点的偏好。申请人的小组最近已经表明，一个相当不寻常的因素成为启动DNA复制在MBT在非洲爪蟾和斑马鱼。这种因子不是蛋白质，而是一种称为Y RNA的非编码RNA。在MBT前，Y RNA的降解对DNA复制没有任何影响，而相反，没有功能性Y RNA的胚胎在MBT后通常不能复制它们的DNA并死亡。本研究项目的目的是了解DNA复制如何在MBT前启动，而不需要Y RNA的功能。来自未发表的试验性实验的结果已经确定，在卵和早期胚胎中存在一种显性起始因子，其以显性方式压倒了DNA复制对Y RNA的要求。该因子的身份尚不清楚，本研究旨在鉴定和表征这种新的主导启动因子。在这个项目中，我们有三个主要目标。(I)洁净.我们将首先通过标准层析技术从非洲爪蟾卵提取物中纯化这种起始因子。我们将监测其在重组无细胞系统中的活性，以启动DNA复制，该系统已耗尽Y RNA功能，因此依赖于这种新的因子。(II)识别.一旦成功地实现了纯化，我们将通过质谱法鉴定主要的起始因子。我们还将确定这个新因子的最接近的人类亲属。(III)表征。该项目将在这里采取两种平行的方式。一方面，我们将阐明该起始因子在非洲爪蟾早期胚胎发育中的正常生物学功能。我们将研究在胚胎发育的关键阶段有多少这种因子存在。我们将分析其活动在关键阶段如何受到监管，特别是围绕MBT。另一方面，我们也将在人类细胞中克隆同源蛋白。我们将研究它是否存在于一组人类细胞中，包括正常细胞、衰老细胞和癌细胞。我们将在缺乏该因子的细胞中表达该因子，并在表达该因子的细胞中关闭其表达，目的是研究该因子是否在正常人体细胞中发挥作用，并研究如果该因子在错误的时间和地点被激活，DNA复制和细胞增殖的后果。