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Various origin mapping approaches have enabled genome-wide identification of origins of replication (ORI) in model organisms, but only a few studies have focused on divergent organisms. By employing three complementary approaches we provide a high-resolution map of ORIs in Plasmodium falciparum, the deadliest human malaria parasite. We profiled the distribution of origin of recognition complex (ORC) binding sites by ChIP-seq of two PfORC subunits and mapped active ORIs using NFS and SNS-seq. We show that ORIs lack sequence specificity but are not randomly distributed, and group in clusters. Licensing is biased towards regions of higher GC content and associated with G-quadruplex forming sequences (G4FS). While strong transcription likely enhances firing, active origins are depleted from transcription start sites. Instead, most accumulate in transcriptionally active gene bodies. Single molecule analysis of nanopore reads containing multiple initiation events, which could have only come from individual nuclei, showed a relationship between the replication fork pace and the distance to the nearest origin. While some similarities were drawn with the canonic eukaryote model, the distribution of ORIs in P. falciparum is likely shaped by unique genomic features such as extreme AT-richness-a product of evolutionary pressure imposed by the parasitic lifestyle., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In Plasmodium falciparum, perinuclear subtelomeric chromatin conveys monoallelic expression of virulence genes. However, proteins that directly bind to chromosome ends are poorly described. Here we identify a novel DNA/RNA-binding protein family that bears homology to the archaeal protein Alba (Acetylation lowers binding affinity). We isolated three of the four PfAlba paralogs as part of a molecular complex that is associated with the P. falciparum-specific TARE6 (Telomere-Associated Repetitive Elements 6) subtelomeric region and showed in electromobility shift assays (EMSAs) that the PfAlbas bind to TARE6 repeats. In early blood stages, the PfAlba proteins were enriched at the nuclear periphery and partially co-localized with PfSir2, a TARE6-associated histone deacetylase linked to the process of antigenic variation. The nuclear location changed at the onset of parasite proliferation (trophozoite-schizont), where the PfAlba proteins were also detectable in the cytoplasm in a punctate pattern. Using single-stranded RNA (ssRNA) probes in EMSAs, we found that PfAlbas bind to ssRNA, albeit with different binding preferences. We demonstrate for the first time in eukaryotes that Alba-like proteins bind to both DNA and RNA and that their intracellular location is developmentally regulated. Discovery of the PfAlbas may provide a link between the previously described subtelomeric non-coding RNA and the regulation of antigenic variation.