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

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 Results from unpublished pilot experiments have established that there is a dominant initiation factor present in eggs and early embryos that overrides the requirement of Y RNAs for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.

In pilot experiments we have discovered a novel dominant DNA replication initiation factor in Xenopus laevis egg extracts. The activity of this factor overrides the requirement of non-coding Y RNAs for the initiation of DNA replication, which is present in somatic vertebrate cells. The activity of this factor can be assayed by its ability to initiate DNA replication in isolated late G1 phase human template nuclei in vitro in the presence of Y RNA-depleted human cell extract.
The fist objective is to purify this initiation factor from Xenopus egg extracts to homogeneity by fast protein liquid chromatography (FPLC).
The second objective is to identify this purified factor by nanoflow LC-MS/MS mass spectrometry. Database searches will allow identification of the closest human homologues. We will obtain antibodies against the Xenopus and human versions of this factor and validate the mass spectrometry results by Western blotting.
The third objective is to functionally characterise this initiation factor. We will initially determine if and how expression, intracellular localisation and activity of this dominant initiation factor are regulated during early development in Xenopus laevis. We will then investigate the role of the human homologue of this initiation factor in the regulation of DNA replication in human cells. If we find that the initiation factor is expressed in some established cell lines, we will deplete it by RNA interference and investigate the consequences on DNA replication and cell proliferation. We will also generate expression constructs for the dominant initiation factor and over-express it in those human cell lines, in which it is not expressed endogenously. We will then analyse by BrdU or EdU pulse-labelling, confocal fluorescence microscopy and flow cytometry if DNA replication is deregulated under these conditions.
In summary, we will characterise a new dominant pathway for the initiation of chromosomal DNA replication in vertebrates.

Who might benefit from this research?
The proposed research is a tightly focused fundamental project in molecular cell and developmental biology. It exploits our recent observation of a novel dominant initiation factor for vertebrate DNA replication and aims to purify, identify and functionally characterise it in vertebrate embryos and human cells. We will be able to meet these goals within the three years allocated for this project. The most obvious and immediate beneficiaries of this research are scientists and their students working in the related fields. The applicant has described this academic impact in the academic beneficiaries section and the case for support of this application. Importantly, however, this research will open up new avenues of subsequent translational work beyond the duration of this project, which has the potential for wider economic and societal impact.
The proposed basic research will lead to the identification of a novel dominant initiation factor for chromosomal DNA replication in vertebrates. It is very likely that the uncontrolled expression of this factor in cells that are not programmed to divide, can lead to untimely replication and cell proliferation. This loss of normal control is a hallmark of cancer. Our basic knowledge of this factor and our understanding of its function in healthy cells and organisms (as generated by this proposed research) will therefore aid, facilitate and guide future translational research to exploit this factor as a novel potential cancer biomarker and target for therapeutic intervention. Therefore, the basic bioscience research proposed here will have an impact on pharmaceutical design and development beyond the duration of this project, and will thus benefit translational scientists, clinicians and their patients.
During this research project, we will also secure potential intellectual property protection and file relevant patent applications where appropriate. The future exploitation of the basic results from this proposed research will include the potential generation of new cancer biomarkers and therapeutic anti-cancer treatments. These developments have clear potential for commercialisation, and potential to attract further R&D investment. Therefore, this work may also benefit the economy.
Cancer is a disease that affects a large proportion of the human population. Any contribution, through basic research, towards the development and improvements of cancer detection and treatment will therefore also have a long-term impact on society by improving health and well-being.

How might they benefit from this research?
The impact of the proposed research outlined above will reach the potential societal and economic beneficiaries indirectly. The first step will be the provision, publication and wider dissemination of our original research results to make them accessible (as detailed in the pathways to impact document). This extension of relevant fundamental biomedical knowledge may then stimulate new research initiatives into further translational and clinical projects, and spin off commercialisation projects exploiting the obtained results.