Investigating the role of arginine methylation as a critical regulator of DNA replication and genome stability

Duplication of the cell's genome is essential for the continuation of life, and this must occur in an efficient and well-timed manner to ensure that each daughter cell gets the correct amount of genetic material. During embryonal development, cells undergo a period of rapid, highly coordinated DNA replication to produce sufficient numbers of cells to sustain growth of the embryo. Consequently, any inherited genetic mutation that slows genome duplication or cell division during this period reduces the total number of cells available for embryonal development, which ultimately reduces the size of the foetus and also certain organs such as the brain. Children born with a genetic disorder that restricts growth and reduces head/brain size are diagnosed with microcephalic primordial dwarfism (MPD).

Whilst MPD is classed as a rare human disease, study of patients with this disease has revealed that a common underlying cause is inherited mutations in genes that encode proteins that function to replicate the genome. From this, it has been hypothesized that a reduction in the efficiency of DNA replication during embryonal development, prevents complete genome duplication being finished in time before the cell divides. This generates damage to the DNA, which triggers the cells with damaged DNA to die. As a result of increased cell death during embryogenesis, overall development of the foetus and several organ systems is restricted, which gives rise to a child with MPD. Therefore, the study of rare human diseases, such as MPD, has dramatically increased our knowledge about how fundamental processes such as DNA replication protect the cell from accumulating genetic damage and the consequences when this process fails. However, despite our progress, we still do not fully understand many important aspects of how these pathways function or indeed whether additional factors exist that are yet to be discovered.

In this respect, we have identified a new protein, DONSON, which is mutated in patients with MPD that functions to help cells replicate their DNA and activate signals when DNA damage is detected. However, whilst it would appear that DONSON gene mutations are a relatively frequent cause of MPD, we still don't understand how DONSON regulates DNA replication, how DONSON signals the presence of DNA damage, how the cell instructs DONSON to carry out its function or why mutations in this gene cause MPD.

Recently, we have identified a critical modification of the DONSON protein, termed arginine methylation. Our preliminary data indicates that arginine methylation of DONSON is vital for its ability to promote DNA replication but our understanding of why this is important is lacking. Therefore, the aim of this proposal is to study how arginine methylation of DONSON controls DNA replication and how defects in this process contribute to the development of disease. We hope by that increasing our knowledge of DONSON function and how it is regulated, our greater understanding of the disease will benefit patients with MPD through better disease management and an improved ability to genetically diagnose it.

Replication stress (RS) is a term that encompasses any type of abnormality that obstructs replication. It is becoming increasingly evident that RS is an important pathological process underpinning the development of human disease. It is thought that the DNA damage caused by incomplete replication leads to increased cell death, particularly in highly proliferative tissues. This is exemplified by Microcephalic Primordial Dwarfism (MPD), which is a genetically heterogenous group of disorders characterised by severe microcephaly, growth retardation and a significantly reduced life expectancy. Whole exome sequencing of MPD patients has identified a common underlying defect in pathways required to maintain genome stability, notably DSB repair and DNA replication. As such, the study of microcephalic disorders and the cellular pathways affected in cells from these patients has significantly furthered our understanding of the molecular mechanisms that have evolved to protect against the deleterious effects of RS. Several years ago, our laboratory identified DONSON as a novel replication factor mutated in patients with MPD. Whilst we demonstrated that DONSON is essential for stabilising a subset of replication forks, in part through its ability to activate ATR, the precise function of this protein and how it is regulated is unknown. Recently, we have demonstrated that DONSON is arginine methylated and that this is essential for unperturbed DNA synthesis. Based on this, we postulate that arginine methylation is a critical regulator of DNA replication. Therefore, this proposal aims to investigate how arginine methylation of DONSON regulates DNA replication and the ATR-dependent replication stress response. From this, our goal is that data generated will not only advance our fundamental knowledge about the post-translational regulation of DNA replication but it will also further our understanding of the mechanisms underlying the development of microcephalic disorders.