The role of DONSON during DNA replication initiation

Our bodies are built of trillions of cells. Over time, our cells age and become damaged, so a subset of cells in our bodies keep growing and dividing, creating their own replacements. Before each cell division, every cell must first duplicate its DNA - all of it, just once and without mistakes. Mistakes during DNA replication that are not timely repaired can lead to mutations and genetic changes that in turn can lead to problems with cell proliferation, aging and development of cancer. Most of the cancer-driving mutations are results of random mistakes during the process of DNA replication. Moreover, hereditary mutations in components of the DNA replication machinery cause a set of disorders characterised by small stature and small brain due to inability to create enough cells to develop a normally sized human being.
To replicate all our DNA is a huge task - we have about 2 metres of DNA in each of our cells, and it is compacted in a highly organised way to fit into the nucleus in a manner that enables proteins to access any needed DNA sequences. During DNA replication this structure must be unwound, duplicated with efficiency and precision, and compacted again. To replicate all DNA, the process of DNA replication starts from about 50 thousand start sites (origins of replication), with some origins being activated early and some late during the process.
The process of origin activation has been well characterised and reconstituted from purified proteins in a simple eukaryotic organism, bakers' yeast, therefore defining the minimal set of proteins needed to fulfil origin activation. However, in more complex organisms, including humans, several players remain unknown. The data we generated in preparation of this proposal suggest that a protein DONSON, which does not exist in yeast, may be a functional equivalent of one of the yeast key origin activators, Sld2, for which such an equivalent in higher eukaryotes is missing. DONSON, when mutated, leads to Meier-Gorlin syndrome - a dwarfism disorder caused by faulty DNA replication initiation; conversely, its overexpression is linked with development of several cancer types. DONSON has been shown to be important for sustaining DNA replication, but its molecular function has not been determined and there is no described role for DONSON in origin activation. Here we propose to investigate the function of DONSON during DNA replication initiation in two higher eukaryotic model systems: cell-free extract prepared from African Clawed frog's eggs and immortalised human cell lines.
We will use biochemical approaches in egg extract to understand where DONSON temporally fits within the origin activation process: which other activators it interacts with, which specific step in the process it plays a role in, and what happens to origins and their activators without DONSON. We will also determine which part of DONSON is important for its function and how it is regulated by enzymes driving origin activation - cyclin dependent kinases (CDKs). All these will establish if DONSON can act as a key activator of replication origins.
In an independent path of investigation, we will determine if DONSON plays a role in origin activation in human immortalised cell lines. We have used genome editing techniques to modify DONSON within cells to fuse it with a degradation tag, which is activated by addition of a plant hormone (auxin) to the cell culture (Auxin Induced Degron, AID). This approach allows for rapid degradation (usually within 30-60 min) of the protein of interest upon auxin addition. We have recently used the AID system combined with cell synchronisation techniques to discover the function of another protein involved in replication - TRAIP. We will now follow an analogous path of investigation with DONSON: we will determine the consequences of DONSON degradation for origin firing using biochemical, microscopy, single-molecule and genome-wide approaches.

Faithful cell division is the basis for the propagation of life and DNA replication must be precisely regulated. DNA replication stress is a prominent endogenous source of genome instability that not only leads to ageing but also neuropathology and cancer development in humans. Specifically, the issues of how vertebrate cells select and activate origins of replication are of importance as, for example, insufficient origin firing leads to genomic instability and mutations in factors involved in origin firing lead to the rare human disease: Meier-Gorlin syndrome. The mechanism of origin firing has been well characterised and reconstituted in yeast, however, equal understanding of this process in higher eukaryotes is lacking.
The firing of replication origins is driven by S-phase kinases (CDKs and DDK) and results in activation of the replicative helicase, CMG, at origins and generation of two bi-directional replication forks. Our preliminary data, generated in the cell-free Xenopus laevis egg extract, show that DONSON is phosphorylated by CDKs and required for CMG formation at origins. Our hypothesis is therefore that DONSON acts as a vertebrate origin activator and possibly as a functional homologue of yeast Sld2. DONSON has been previously shown to be essential during DNA replication, both in human cells and in Drosophila, but the mechanism of DONSON's action is unknown. It was shown to interact with the replisome and was proposed to protect stalled DNA replication forks, but its potential role in replication initiation was never investigated.
This project is aimed at delivering the molecular function of DONSON, especially during replication initiation. We will use a combination of the X.l. egg extract system and rapid degradation of DONSON in human immortalised cells to deliver the first in-depth understanding of DONSON's interactions, functions and regulation in DNA replication.