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

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 Results from unpublished pilot experiments have established that there is a dominant initiation factor present in eggs and early embryos that overrides the requirement of Y RNAs for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