How do common and diverged features of the replicative stress response shape the biology of TriTryp parasites?

The growth and propagation of any living organism is dependent on the faithful transmission of its genome - the genetic material that provides the blueprint for life. Transmission of the genome to offspring requires that the genetic material is copied and then segregated. The copying process is referred to as replication. During replication, the complex machinery that directs this process encounters many obstacles, which further cellular machinery both recognises and then resolves, allowing restart of the replication process. One pronounced obstacle to replication is the cell's machinery that allows expression of the genes in the genome, and it is known that clashes between gene expression and replication can result in genome changes. We are interested in these clashes in a set of parasites termed the Kinetoplastids, which have been the subject of extensive study, at least in part because they display numerous aspects of their biology that are unconventional relative to other eukaryotic cells and because they cause pronounced human suffering and economic hardship world-wide. Previous work has suggested that gene expression, despite being one of the most central processes in any cell, is unconventional in all kinetoplastids. In addition, our recent work has shown that genome replication, an equally central cellular reaction, appears to differ between the kinetoplastid parasites in which it has been studied. These findings suggest to us two things, which we wish to test. First, we hypothesise that all kinetoplastids share a common problem of elevated levels of clashes between gene expression and replication, necessitating robust and perhaps novels means to tackle this type of obstacle to allow the completion of genome copying. Second, we hypothesise that the means that are adopted to tackle these collisions are subtly different in the three kinetoplastids, resulting in different forms of variation in their genomes, which lead to the very different interactions that they display with the humans and other mammals that they infect. By testing these hypotheses, we believe that we will reveal differences or similarities between kinetoplastids and other eukaryotes in the strategies used during genome replication, which may also reveal pathways to target the parasites and alleviate the diseases they cause.

Kinetoplastid parasites cause disease worldwide and share a common, diverged genome organisation in which virtually all their genes are expressed from multigenic clusters. Each cluster has a single promoter, meaning the need for transcription to traverse many genes must result in frequent and problematic clashes between transcription and replication, a pronounced replicative stress that has been little explored. Despite this commonality, kinetoplastid parasites have evolved substantial differences in life cycles and infection strategies. Moreover, genome architecture and replication dynamics vary widely in Trypanosoma brucei, T. cruzi and Leishmania. We propose that differing responses to replicative stress, most commonly transcription-replication clashes, explain the differing genome biologies. To test this hypothesis we propose to (1) map the genomic sites of replication-transcription clashes in the three parasites; (2) determine the genome-wide responses to replicative stress; (3) characterize how the three parasites sense and signal replicative stress; and (4) compare the functions of predicted factors that mediate recovery from replicative stress. We suggest that in pursuing these questions we will describe critical genome maintenance functions that drive adaptive changes in gene and chromosome content, which have been associated with drug resistance and underlie the differing strategies for parasite growth and survival. Genome content changes in response to repliciaton stress are not limited to the tritryp parasites, and so the processes that we describe will have signifcance for all organisms, including diseases in humans that can result from genome alterations, such as cancer.

The immediate beneficiaries of this work will be academic researchers in the fields of parasitology and genome biology (DNA replication and repair, and in cell cycle control). This impact will result from the fundamental findings that emerge from the characterisation of how the replication stress response in the tritryp parasites contributes to variations in genome architecture and stability. Our work will have wider relevance for such processes in other parasites, in other microbes and in all organisms, including how DNA replication is orchestrated genome-wide, how copy number variation emerges in genomes and how genome adaptions contribute to variations in life and cell cycles. We will deliver extensive, novel data sets that will allow comparisons to be made between the three parasites, and to the wider fields of genome and pathogen biology. Our work may reveal aspects of how drug resistance arises in the tritryp parasites and, if so, we will share this knowledge with relevant organisations (e.g. the Drugs for Neglected Diseases Initiative, the World Health Organisation), thus contributing to the health and wellbeing of the countries affected by these parasites. The work on this project falls within the broad area of microbial biology, which has a wide impact on the health of the population of the UK and beyond, and is frequently the subject of media discussion (e.g. through television programmes, such as Horizon or 'Monsters Inside Me', and in public science exhibits, such as at the Glasgow Science Centre). We will contribute to this discussion, throughout the course of the project, through public lectures and exhibits, school outreach programs and articles in local and national media.