Investigating how replication fork rotation causes chromosomal instability during S phase

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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.