ATR, a DNA damage kinase, modulates DNA replication timing in Leishmania major.

All cells possess mechanisms to maintain and replicate their genomes, whose integrity and transmission are constantly challenged by DNA damage and replication impediments. In eukaryotes, the protein kinase Ataxia-Telangiectasia and Rad3-related (ATR), a member of the phosphatidylinositol 3-kinase-like family, ensures genome maintenance and stability, and is considered as a master regulator of the eukaryotic response to DNA injuries. Here we aimed to investigate the conservation and functional relevance of the ATR homolog in the DNA metabolism of Leishmania major, a protozoan parasite with a remarkably plastic genome. CRISPR/cas9 genome editing was used to generate a Myc-tagged ATR cell line (mycATR), and a Myc-tagged C-terminal knockout of ATR (mycATR?C-/-). We show that ATR nuclear localisation depends upon the C-terminus and its mutation results in accumulation of single-stranded DNA, impaired cell cycle control, increased levels of DNA damage, and delays DNA replication re-start after replication stress. In addition, we show that ATR is a key player for the maintenance of L. major's unusual DNA replication program, where larger chromosomes are replicated later than smaller chromosomes, and reveal that loss of this program in ATR mutants is related to the accumulation of replication signal around replicative stress fragile sites, which are enriched in larger chromosomes. Finally, we show that such changes in the DNA replication program leads to chromosome instability and affects genome variability. In summary, our work shows that ATR acts to connect DNA replication timing and variability of the Leishmania genome.