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Does genome replication in Leishmania rely on origin-independent initiation?

Reference	BB/R017166/1
Principal Investigator / Supervisor	Professor Richard McCulloch
Co-Investigators / Co-Supervisors
Institution	University of Glasgow
Department	College of Medical, Veterinary, Life Sci
Funding type	Research
Value (£)	474,072
Status	Completed
Type	Research Grant
Start date	01/12/2018
End date	30/11/2022
Duration	48 months

Abstract

The orthodox view of genome replication is that the binding of conserved replication initiator factors to defined genome sites, termed origins, is an essential first step in the DNA synthesis process. However, this view has been questioned by studies, in both prokaryotes and eukaryotes, in which has been shown that genome replication can proceed in cells in which origins or initiators have been experimentally ablated or impaired. These findings suggest greater flexibility in replication initiation strategies than current textbook descriptions. However, no organism has been described to date that relies on origin/initiator-independent DNA replication as a normal component of genome copying. Published data suggest that Leishmania may be such an organism. We propose Leishmania employs a bimodal strategy for replicating its chromosomes in which constitutive initiation at a single locus-specific origin is supported by stochastic, locus-unspecific replication initiation. This project will test this prediction, addressing three hypotheses that underlie the bimodal replication proposal: (1) replication initiation at the single conventional origin in each Leishmania chromosome is due to interdependence of centromere and origin functions due to co-localization of the kinetochore and initiator machineries; (2) replication initiation in each chromosome also involves hundreds of infrequent, flexible origin/initiator-independent events; (3) origin/initiator-independent replication initiation relies upon recombination-directed events, which leads to the known, pervasive genome plasticity of these parasites. By testing these hypotheses, the project will address the evolution of origin multiplicity in eukaryotes, the role of replication flexibility in driving genome variation and rearrangement, and whether novel aspects of Leishmania replication might be future directions for therapeutic intervention, such as through inhibition of recombination.

Summary

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 from parent to offspring requires that the genetic material is copied and then segregated. The copying process is referred to as replication. In cellular organisms, the genome is made of DNA and its replication is normally described as a highly regulated process in which specific proteins recognise and bind to specific regions in the genome, termed origins, where copying of the genome begins. In bacteria one origin is found in the genome, whereas more complex organisms, the eukaryotes, which include humans, possess hundreds or thousands of origins in each of the chromosomes that make up their genome. Despite this textbook description of origins being essential for replication, some studies have shown that genome replication can occur when origins are removed, or when the origin binding proteins are deleted or their function disturbed. A key question, then, is whether or not some cellular organisms might normally replicate their genomes without relying on origins. If so, the description of origin-independent replication would alter our view of the evolution and adaptability of genome replication strategies. In this project, we wish to determine if Leishmania, a parasite that causes leishmaniasis, a devastating disease worldwide, might be the first example of a eukaryotic organism in which genome copying relies on flexible, origin-independent replication initiation events, in addition to more limited origin-defined replication. We will pursue cutting-edge genetic and genome-wide analyses to test our prediction of an unusual, bimodal strategy for DNA replication in Leishmania chromosomes, including how replication might be stimulated at non-origin sites of the genome. We suggest that our hypothesis of Leishmania reliance on origin-independent replication explains the known propensity of this parasite, and its relatives, to rapidly vary the genetic composition of their genome, which might in the future lead to new therapies against the diseases they cause and to the better understanding of their pathogenicity and the development of resistance mechanisms to drugs. In addition, our work might explain genome variation in wider organisms, including where genome rearrangement leads to disease, for e.g. cancer, and developmental disorders.

Impact Summary

The immediate beneficiaries of this work will be academic researchers in the fields of parasitology and genome biology (DNA replication and repair). Impact will result from the fundamental findings that emerge from the characterisation of how Leishmania parasites replicate their genome, and how this 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 plasticity contributes to variations in life and cell cycles. We will deliver extensive, novel data sets that will allow comparisons to be made with related kinetoplastid parasites, and to the wider fields of genome and pathogen biology. Our work may reveal aspects of how drug resistance arises in Leishmania 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, 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. In addition to having academic impact, we envisage further benefits: Economic benefits. This work is concerned with the study of a fundamental process in cell growth and development and is not, therefore, likely to have direct economic benefits, such a through the identification and development of novel therapeutic compoundsagainst kinetoplastid parasites. Nonetheless, the funds provided will contribute to the local communities in which is dispersed, and the training of the researchers, hopefully leading to careers in science, will have long term economic benefits in the UK and beyond. Societal benefits. This project falls within the broad subjects of genetics and microbial biology, with relevance to the health of the population of the UK and beyond. To disseminate this knowledge, the Principal Investigator and the researchers will contribute to the 'public understanding of science', through public lectures, school outreach programs and contribution to local and national media. In doing so, we will promote access to the science beyond the confines of academic institution(s).

Committee	Research Committee C (Genes, development and STEM approaches to biology)
Research Topics	Microbiology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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