Investigating how replication fork rotation causes chromosomal instability during S phase

DNA is the information storage material of our cells. It is composed of two very long intertwined polymers, each made up of four distinct nucleotides. The sequence of these nucleotides together encodes the overall blueprint, or genetic code of the functioning cell. To ensure the blueprint is maintained every time a cell divides the DNA polymers have to untangled from one another and exactly duplicated. This remarkable feat is achieved by a collection of enzymes collectively known as the DNA replication machinery. It is estimated that the DNA replication machinery normally hardly ever makes a mistake. However, this fidelity is diminished in cancer cells and human diseases that induce premature aging. In these cells the nucleotide sequence often changes and the chromosomes are frequently broken and rejoined. However, the sites of breakage are not random. Instead, breakage often occurs in distinct areas commonly termed "fragile sites". At fragile sites it is thought that the chromosomes are especially difficult to separate and copy, leading to a errors and breakage.

These errors can irreversibly change the behavior of cells by mutating the genome. This can cause cells to senesce, causing ageing or promote the development of cancer. Occasionally defects in DNA replication are found to be associated with rare human developmental disorders such as microcephaly. Therefore errors during DNA replication can have both widespread and specific effects. Why this is so is unknown, however it seems likely that distinct problems during replication affect fragile sites differently, leading to variable outcomes.

Our understanding of why replication appears to be error prone at fragile sites has been greatly aided through studies of relatively simple eukaryotic cells, such as yeast that replicate DNA in a very similar fashion to human cells. These have shown that genomic sites where the replication machinery collides with other processes working on DNA are often "fragile" with increased DNA damage appearing to occur around them. These studies have contributed to the idea that when the replication machinery encounters other processes, the error rate dramatically increases. However, how this occurs is unknown.

In our recently submitted work we have found a wholly novel explanation for errors and damage occurring at fragile sites. We have found that problems in untangling the DNA can lead to "braiding" of the replicating DNA. This leads to problems in duplicating the unwound strands, causing DNA damage in the newly replicated DNA. Damage caused by this pathway is closely linked to replication through candidate yeast fragile sites.

In this proposal we wish to extend this analysis to define the yeast and human fragile sites where this novel pathway to DNA replication associated DNA damage is acting. We will then assess the types of mutations that are likely to be caused by this pathway and link these to the different cellular problems that DNA replication can induce. To do this we will use techniques where DNA damage can be quantified across an entire genome and assess when DNA damage is caused specifically under conditions that amplify the damage caused by the novel pathway through braiding of the DNA. We will then use this data to carefully describe the conditions that lead to chromosome fragility through the novel pathway.

In many ways the multiple rounds of DNA replication that occur over our lifetime are the crucial difference between the ageing cells in our bodies and the "ageless cells" of our gametes. Therefore understanding where and why DNA replication changes our genetic code, changing cellular function, is a crucial step to understanding ageing and potentially counteracting the biological aspects of ageing most problematic for modern society.

DNA replication stress is a major source of genomic instability and has been linked to ageing of stem cell niches, cancer evolution and developmental abnormalities. DNA damage due to replication stress varies according to genomic context but the reasons for this are poorly understood. One type of replication stress occurs at locations that pause or arrest replication and DNA damage is frequently observed at such sites. Therefore these loci are termed fragile sites. Our recent work has outlined a novel pathway to generating DNA damage at certain fragile sites. We have found that stable protein-DNA sites that pause replication cause the replication fork to rotate in order to unwind the DNA. Increased fork rotation causes DNA catenation, which appears to impede processes that occur behind the fork. This results in DNA damage in the resultant sister chromosomes.

In this proposal we aim to define where and why replication fork rotation leads to DNA damage and if fork rotation is a defining feature of fragile sites. This analysis will be carried out primarily in yeast cells, although we will also examine human cells to investigate evolutionarily conserved features of this pathway. By examining the genetic modifiers and DNA repair pathways utilized at the sites of topoisomerase II associated damage we will define whether the damage incurred is due to fork rotation or to other, distinct, pathways of chromosome stability. We will then go onto examine how the DNA damage generated by fork rotation is repaired and contrast this with the repair pathways required following other types of replicative stress. We aim to fully describe how the varying context of DNA replication cause different types of stress and genome instability and then go on to link these events with the pleiotropic downstream consequences of replication stress on human health.

This proposal investigates how topological stress can lead to permanent changes in cell behavior. We have recently described a new pathway of replication stress whereby local topological stress leads to the accumulation of DNA damage at known fragile sites in yeast. In this proposal we aim to demonstrate the genome wide relevance of this pathway in both yeast and human cells.

In the short term the primary beneficiaries of this research will be academics interested in the mechanisms of replication stress. The effects of topological stress on DNA replication are poorly understood. It is crucial for research into this field that the genome-wide importance of this novel pathway is investigated and disseminated to the rest of the field as soon as possible. This research will also link heritable mutation caused by topological stress to downstream changes in cell behavior. Therefore it will have widespread long-term impact not only in the replication field but also potentially in other fields relating genetic changes to human disease.

In the longer term our research is potentially important for clinicians attempting to counteract human conditions where replication stress is thought to be a root cause. DNA replication stress is linked with ageing, cancer and abnormal development. However the underlying physiology of the linkage is unclear. We will describe the genomic contexts where topological stress damages DNA and also the range of heritable lesions likely to be incurred in these contexts. This will provide crucial molecular pathways for clinicians treating ageing, cancer and abnormal development to reference. In the long term it will ultimately lead to novel regimens and enhancing the quality of life for this group of patients.

Several chemotherapies act by "poisoning" topoisomerase activity. Topoisomerases relax topological stress and are therefore crucial regulators of topological effects. So although the main killing effect of these agents is from un-repairable DNA breakage in cells, it is also likely that they have other, potentially locus specific, effects through increasing the levels of topological stress in the cells. Our work will provide data on how the induced topological stress could contribute to toxicity. We will investigate the potential effects of increased topological stress and the downstream mutational consequences of this type of stress. Through subsequent projects built on this work we aim to aid clinicians in designing new regimens to maximize their cancer killing efficacy, while minimizing their unwanted effects in normal cells.

This project will also have considerable capacity building impact both for the PDRA recruited and for the GDSC and the wider Department of Life Sciences at the University of Sussex. Biological research is increasingly becoming a data driven research area. Strategically the GDSC is focused on producing researcher with the skills to produce both the highest quality data and the highest quality analysis of that data. In this proposal the PDRA we will be trained in both these areas internally and externally, gaining crucial skills for use in the U.K. research arena. Through interaction with the other GDSC researchers involved in data driven research they will also enhance the effectiveness of the GDSC as a whole. In addition through interaction with researchers, both nationally and internationally they will hopefully improve research across both the national and international research community.

This training and experience will be potentially of great benefit to the PDRA and the wider U.K. economy. In the wider economy the benefits of data driven research to improve productivity in all processes is clear. The PDRA will have the ability to evaluate and analyse metadata from potentially any sector to identify significant correlation and potentially identify new work processes.