Abstract
DNA replication is needed to duplicate a cell's genome in S phase and segregate it during cell division. Previous work in Leishmania detected DNA replication initiation at just a single region in each chromosome, an organisation predicted to be insufficient for complete genome duplication within S phase. Here, we show that acetylated histone H3 (AcH3), base J and a kinetochore factor co-localise in each chromosome at only a single locus, which corresponds with previously mapped DNA replication initiation regions and is demarcated by localised G/T skew and G4 patterns. In addition, we describe previously undetected subtelomeric DNA replication in G2/M and G1-phase-enriched cells. Finally, we show that subtelomeric DNA replication, unlike chromosome-internal DNA replication, is sensitive to hydroxyurea and dependent on 9-1-1 activity. These findings indicate that Leishmania's genome duplication programme employs subtelomeric DNA replication initiation, possibly extending beyond S phase, to support predominantly chromosome-internal DNA replication initiation within S phase.
Highlights
Once every cell cycle, a cell must completely duplicate its genome before segregating the two resulting copies into offspring cells
Previous Marker Frequency Analysis coupled with deep sequencing (MFA-seq) analysis, which detected a single region of replication initiation per chromosome in L. major and L. mexicana (Marques et al, 2015), is potentially limiting because it relies on cell sorting to enrich for S phase and G2 or G1 cells, inferring regions of DNA synthesis by comparing sequence read depth in the former relative to the latter
We reasoned that L. major in stationary phase could serve as a non-replicative control for normalisation in MFA-seq analysis compared with cells that are growing exponentially
Summary
A cell must completely duplicate its genome before segregating the two resulting copies into offspring cells. Genome duplication relies on DNA replication, a normally tightly controlled and high-fidelity reaction (Burgers and Kunkel, 2017). DNA replication is initiated at genomic loci termed origins, which are sequence-conserved features in prokaryotes (Leonard and Mechali, 2013). With the exception of Saccharomyces and closely related yeasts (Dhar et al, 2012), replication origins in eukaryotes are not defined by conserved sequences. More elusive features, such as chromatin accessibility, transcription level and epigenetic elements (MacAlpine et al, 2010; Cayrou et al, 2015; Deal et al, 2010; Dellino et al, 2013; Lombrana et al, 2013; Mesner et al, 2011; Chen et al, 2019), are determinants of replication initiation activity. At the onset of S-phase origins are fired, initiating DNA synthesis that proceeds bi-directionally along the chromosomes
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