Collectively, we have advanced the state of the art in our knowledge of Plasmodium DNA replication. We have produced and published a suite of high-resolution tools that can now be used for mapping and quantifying replication under any condition, such as antimalarial drug pressure. We have also produced a highly-cited review, a well-received hypothesis article, and a special journal collection with associated editorial, all promoting this fast-growing field of research.
In Aim 1, we followed DNA replication through schizogony and gametogenesis with a time-resolution that was completely unprecedented in the literature. A colleague has since developed continuous live-cell imaging (as opposed to timepoint-series of fixed cells), improving time-resolution still further, albeit at the expense of spatial resolution. We are now working together to exploit these complementary techniques. Others in the field are exploiting high-resolution microscopy such as FIB-SEM. Overall, the state of the art in this area is advancing rapidly. In particular, our collective understanding of the fast, poorly-regulated process of gametogenesis has greatly improved. This represents a potential ‘Achilles heel’ for the parasite – vulnerable to replication-damaging interventions such as drugs, and crucial for the transmission of malaria.
In Aims 2 and 3, DNA replication had never previously been examined at single-molecule resolution in Plasmodium, nor in any organism with such a highly A/T-biased genome. All our data on this topic are novel and, thus far, unique. The technique we developed (single-molecule replication mapping via nanopore sequencing) laid the groundwork in understanding the landscape of replication origins & replication fork speed in Plasmodium. It has great potential for high-resolution studies of the acute effects of antimalarial drug treatment, which may stall replication forks. It also has wider potential in fields such as cancer research. In terms of basic biology, Plasmodium also offered us a unique opportunity to compare replication origin activity in two genomes with similar cell cycles but vastly different A/T contents (P. falciparum & P. knowlesi) – this is yielding fundamental insights about replication origin determination in ongoing work within Aim 3.
Wider societal impacts have not occurred thus far, as expected from a project focussed on basic-biology and technique development, but there are real prospects for future impact. These are in guiding development of synergistic antimalarial drugs and/or anti-transmission drugs.
