Origins of DNA replication in Leishmania mapped by DNAseq or cell-cycle sorted promastigotes

The earliest stage of DNA replication is the designation of defined genome sites, termed origins, where DNA synthesis initiates. Origins are binding sites for replication initiator factors, which mediate recruitment of the replication machinery (1). Despite the fundamental role of DNA replication in life, origin structure and usage is not conserved across biology (2). The genomes of most bacteria and many archaea are replicated from single origins, which fire in every replication cycle, display sequence conservation within each kingdom and, at least in bacteria, are largely conserved in genome position (3). In contrast, in every eukaryote examined to date each linear chromosome is replicated from multiple origins that display variations in frequency and timing of firing. Moreover, identification of a consensus origin sequence amongst the multiple mapped sites has proved impossible in nearly all eukaryotes, with the exception of Saccharomyces yeast and its relatives (4). Here we map replication origins genome-wide in Leishmania and show that origin usage in this kinetoplastid parasite does not conform to eukaryotic orthodoxy: each chromosome contains only a single detectable origin, and all origins act with similar efficiency. Origin singularity appears fundamental to the replication of each Leishmania chromosome, since comparing two species shows that this strategy is maintained during evolutionary changes in which chromosome fusions or fissions have occurred. Origin usage in Leishmania is distinct even from the related parasite Trypanosoma brucei, whose chromosomes employ multiple origins for replication (5). Despite this, we show that many origins in the two parasites are positionally conserved, though only in Leishmania can we identify origin-specific structural features. Our study reveals highly unorthodox origin usage in a microbial eukaryote, which has implications for genome maintenance and the evolution of origin multiplicity and associated controls.