Leishmania major chromosomes are replicated from a single high-efficiency locus supplemented by thousands of lower...

DNA replication initiates at genome sites termed origins. Previous origin mapping approaches in the populations of the protozoan parasite Leishmania major were discordant, suggesting either a single origin per chromosome or 200-fold more origins. To reconcile these data and fully understand DNA replication dynamics, we have applied DNAscent, an assay that detects patterns of 5-bromodeoxyuridine (BrdU) incorporation in individual long-read DNA molecules. We confirm the pre-eminence of a single locus of DNA replication initiation in each chromosome and reveal a much larger number of lower-efficiency DNA replication initiation events whose abundance is greater as chromosome size increases. Each initiation site is a region of high AT content, increased G-quadruplex levels, lowered chromatin occupancy, and reduced levels of nascent RNA. Finally, we show that all DNA replication initiation results in mutagenesis. This work reveals a bimodal strategy for DNA replication programming in Leishmania that drives replication timing and sequence variation.