Luiz Ricardo Orsini Tosi

Genome plasticity, manifested as gene copy-number variation and mosaic aneuploidy, enables adaptation in Leishmania , yet how genome-surveillance pathways shape this variation remains unclear. Here we show that partial depletion of HUS1 or RAD9, subunits of the conserved 9-1-1 checkpoint clamp, produces opposing replication-stress responses and contrasting patterns of genome remodelling in Leishmania major . During methotrexate selection HUS1 deficiency restricted DHFR-TS amplification and favoured linear amplicons, whereas RAD9 deficiency was associated with extensive circular amplification. This divergence extended to genome-wide scale: HUS1-deficient cells carried fewer but larger candidate rearrangements, whereas RAD9-deficient cells retained a largely wild-type rearrangement burden. These differences were accompanied by distinct HUS1 and RAD9 occupancy profiles around repetitive DNA implicated in gene amplification. Deficiency of either of these factors also resulted in differing effects on aneuploidy. While HUS1-deficient cells showed limited shifts towards chromosome disomy, RAD9-deficient cells exhibited broader chromosome copy-number gains and losses. Together, these findings identify replication-stress surveillance as a determinant of parasite genome plasticity, revealing that perturbing individual subunits of the same checkpoint complex can produce opposing genome-remodelling outcomes and reshape the variation available for adaptation.

Exposed single-stranded DNA (ssDNA) accumulating at stalled or collapsed replication forks (RFs) can trigger the replication stress response (RSR) to prevent genome instability. Leishmania are parasitic eukaryotes whose plastic genomes facilitate rapid adaptations under stress, with intrinsic replication stress (RS) implicated as a source. Little is known about the Leishmania RSR. Here, we reveal the global dynamics of the RSR in Leishmania major by performing ChIP-seq, focusing on two key RSR proteins, γH2A and RPA1, in the absence and presence of RS. We show that common RS ‘hotspots’ coincide with DNA replication initiation and transcription termination in Leishmania. When replication is stalled, RSR factors accumulate at early S-phase origins, with a signal pattern reminiscent of bidirectional RF progression. Under chronic RS conditions, increased accumulation of RSR factors emerges at wider sites of transcription initiation, suggesting Leishmania may possess compensatory strategies to limit the effects of RS and ensure DNA replication completion under stress. In contrast, chronic RS enhances RSR factor accumulation at transcription termination sites, highlighting these regions as RS ‘hotspots’ in Leishmania. Lastly, variations in replication protein A (RPA) dynamics in Ataxia Telangiectasia and Rad3 Related (ATR)-deficient cells uncover crucial roles of this kinase in managing polycistronic transcription and DNA replication, particularly under RS, in Leishmania.

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, acts as a master regulator of the eukaryotic response to DNA injuries, ensuring DNA replication completion and genome stability. Here we aimed to investigate the 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 (mycATRΔC-/-). We show that the nuclear localisation of ATR depends upon its C-terminus. Moreover, its deletion results in single-stranded DNA accumulation, impaired cell cycle control, increased levels of DNA damage, and delayed DNA replication re-start after replication stress. In addition, we show that ATR plays a key role in maintaining L. major’s unusual DNA replication program, where larger chromosomes duplicate later than smaller chromosomes. Our data reveals loss of the ATR C-terminus promotes the accumulation of DNA replication signal around replicative stress fragile sites, which are enriched in larger chromosomes. Finally, we show that these alterations to the DNA replication program promote chromosome instability. In summary, our work shows that ATR acts to modulate DNA replication timing, limiting the plasticity of the Leishmania genome.

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 acts as a master regulator of the eukaryotic response to DNA injuries, ensuring DNA replication completion and genome stability. Here we aimed to investigate the 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 (mycATRΔC-/-). We show that the nuclear localisation of ATR depends upon its C-terminus. Moreover, its deletion results in single-stranded DNA accumulation, impaired cell cycle control, increased levels of DNA damage, and delayed DNA replication restart after replication stress. In addition, we show that ATR plays a key role in maintaining L. major’s unusual DNA replication program, where larger chromosomes duplicate later than smaller chromosomes. Our data reveals loss of the ATR C-terminus promotes the accumulation of replication signal around replicative stress fragile sites, which are enriched in larger chromosomes. Finally, we show that these alterations to the DNA replication program promote chromosome instability. In summary, our work shows that ATR acts to moderate DNA replication timing thus limiting the plasticity of the Leishmania genome.

When exposed single-stranded DNA accumulates at stalled or collapsed replication forks, the replication stress response is triggered to prevent genome instability. Leishmania are parasitic eukaryotes where gene expression is universally polycistronic and whose plastic genomes facilitate rapid adaptations in response to stress, with evidence implicating intrinsic replication stress as a source. Little is known about the Leishmania replication stress response. In this study, we reveal the global dynamics of the replication stress response in L. major promastigotes by performing ChIP-seq on three key replication stress response proteins, γH2A, RPA1 and RAD9, in the absence and presence of replication stress. We show that common ‘hotspots’ of replication stress correlate with DNA replication initiation and transcription termination in Leishmania . When DNA replication is stalled, replication stress response factors accumulate at early S-phase origins, with a signal pattern reminiscent of bidirectional replication fork progression. Under conditions of chronic replication stress, increased accumulation of replication stress response factors emerges at wider sites of transcription initiation, suggesting Leishmania may possess compensatory strategies to limit the effects of replication stress and ensure DNA replication can complete under these conditions. In contrast, chronic replication stress enhances RSR factor accumulation at transcription termination sites, highlighting these regions as key replication stress ‘hotspots’ in Leishmania . Lastly, variations in RPA dynamics in ATR-deficient cells uncover crucial roles of this protein kinase in managing polycistronic transcription and DNA replication, particularly under replication stress, in Leishmania . Summary: Strict controls operate to precisely copy an organism’s DNA. However, cells need ways to rapidly adapt and respond to stimuli. In some cases, these beneficial adaptations come from problems during replication. Leishmania parasites cause serious neglected infections in humans and animals across the world’s tropics and sub-tropics. Remarkably, recent evidence suggests that Leishmania DNA experiences enhanced stress during replication that can drive its ability to rapidly adapt in response to stress. How L eishmania respond to DNA replication stress is still poorly understood. Here, using a genome-wide approach to map the locations of key proteins that manage DNA replication stress and maintain genome integrity, we show ‘hotspots’ of DNA replication stress coincide with start sites of DNA replication and regions of transcription termination.

Eukaryotic chromosomes segregate faithfully prior to nuclear division to ensure genome stability. If segregation becomes defective, the chromosome copy number of the cell may alter leading to aneuploidy and/or polyploidy, both common hallmarks of cancers. In eukaryotes, aurora kinases regulate chromosome segregation during mitosis and meiosis, but their functions in the divergent, single-celled eukaryotic pathogen Trypanosoma brucei are less understood. Here, we focused on one of three aurora kinases in these parasites, TbAUK3, a homologue of the human aurora kinase AURKC, whose functions are primarily restricted to meiosis. We show that RNAi targeted depletion of TbAUK3 correlates with nuclear segregation defects, reduced proliferation, and decreased DNA synthesis, suggestive of a role for TbAUK3 during mitotic, not meiotic, chromosome segregation. Moreover, we uncover a putative role for TbAUK3 during the parasite’s response to DNA damage since we show that depletion of TbAUK3 enhances DNA instability and sensitivity to genotoxic agents. HIGHLIGHTS: The C-terminus of TbAUK3 is disordered TbAUK3 depletion coincides with nuclear segregation defects Depletion of TbAUK3 enhances DNA instability

Editorial: Leishmania genome variability: Impacts on parasite evolution, parasitism and leishmaniases control