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

一个健康的多细胞有机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，对人类和动物的健康至关重要的是，潜在的分子机制到位，确保DNA复制只在正确的时间和地点发生。这项研究的目的是描述一种在脊椎动物早期发育过程中调节DNA复制的新的分子机制。在脊椎动物的早期发育过程中，DNA复制的调节发生了巨大的变化。对这一规定理解得最好的模式生物是非洲爪蛙，非洲爪蛙。非洲爪哇卵受精后，在12个短的分裂周期内快速复制其DNA，没有任何选择从哪里开始。事实上，DNA复制已经在有丝分裂的个别染色体上开始了。从激活的鸡蛋中提取的提取物重述了这一规则，并非常迅速地复制了实验中添加的任何DNA。在最初的12个短分裂周期之后，胚胎经历了一个过渡(称为中期囊胚过渡，或MBT)，此时DNA复制变得更加严格。在细胞分裂周期中，DNA复制与有丝分裂是由不同的间隙阶段分开的，它始于对特定染色体位置的偏好。申请人的团队最近表明，在非洲爪哇和斑马鱼的MBT处，一种相当不寻常的因素成为启动DNA复制的关键因素。这个因子不是一种蛋白质，而是一种称为Y RNA的非编码RNA。Y RNA的降解对MBT前的DNA复制没有任何影响，而相反，缺乏功能Y RNA的胚胎在MBT后无法复制DNA并死亡。本研究的目的是了解DNA复制如何在MBT之前启动而不需要Y RNA功能。未发表的初步实验结果表明，卵子和早期胚胎中存在一个显性的启动因子，它以一种显性的方式取代了Y RNA对DNA复制的要求。这个因素的身份尚不清楚。这里提出的研究目的是识别和表征这种新的和主导的启动因素。在这个项目中，我们有三个主要目标。(I)净化。我们将首先用标准的层析技术从非洲爪蛙卵提取液中纯化这种启动因子。我们将在一个重组的无细胞系统中监测它的活性，以启动DNA复制，该复制已经耗尽了Y RNA功能，因此依赖于这个新的因子。(Ii)识别。一旦纯化成功，我们将通过质谱学鉴定主要的引发因素。我们还将确定这一新因素的最接近的人类亲属。(Iii)定性。在这里，这个项目将分两条路走。一方面，我们将表征这种启动因子在非洲爪哇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一因素存在。我们将分析在关键阶段如何监管其活动，特别是围绕MBT。另一方面，我们还将描述人类细胞中的同源蛋白质。我们将调查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，对DNA复制和细胞增殖的影响。

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.