Understanding how DNA replication programming drives adaptive genome variation in African trypanosomes and Leishmania
Professor Richard McCulloch, School of Infection and Immunity
DNA replication is a central reaction in life, providing for genome inheritance and maintenance of cell function during growth and development. To ensure accurate genome transmission, eukaryotic cells have evolved tightly controlled programmes of DNA replication in which new copies of the genome are generated from multiple loci termed origins, where DNA synthesis initiates during S-phase of the cell cycle. Origins are defined by binding of an initiator complex, termed the Origin Recognition Complex (ORC), leading to a cascade of replication factor recruitment and activation. Despite considerable mechanistic understanding of these events, many questions remain: what features dictate origin location and function; how flexible is the origin-driven programme of DNA replication; and when can cells employ origin-independent DNA replication? Answering these questions is critical to understand how genome content evolves, either incrementally during evolution, or abruptly to adapt cell behaviour in response to environmental change.
An understanding of eukaryotic DNA replication is largely derived from work in a limited number of organisms, including yeast, mammals and Drosophila, and findings have been generalised across this diverse domain of life. This application seeks to build upon work in two important eukaryotic parasites, Trypanosoma brucei and Leishmania, which has suggested divergence in the DNA replication programme and machinery relative to other characterised eukaryotes. A major potential deviation from other eukaryotes derives from whole genome mapping analyses, which suggest these parasites replicate their genomes with insufficient numbers of ORC-defined origins to complete replication of their complete genomes during S-phase. If so, such origin paucity may indicate that DNA replication flexibility in central to the biology of these parasites, perhaps allowing elevated rates of genome sequence change. Such sequence change may be crucial for survival and transmission, such as by antigenic variation.
This project will seek to test the predicted organisation of DNA replication in the two parasites. To do so, next generation sequencing strategies, some using long-read Nanopore approaches, will be deployed to detect and characterise all locations of DNA replication in single cells, as well as to look at patterns of DNA synthesis, including rate, pausing and termination. In addition, the project will engineer the genomes of the two parasites using CRISPR and site-specific recombination to change the number of origins, as well as move origins from known locations into putative origin-free genome regions, thereby testing the constraints of DNA replication programming.
The project will involve molecular and cellular biology, genetic modification and culture of parasites, and bioinformatics. A recent review of this topic from our lab is below:
Damasceno, J. D., et al. (2021). "Read, Write, Adapt: Challenges and Opportunities during Kinetoplastid Genome Replication." Trends Genet 37(1): 21-34.