Replication-stress response shapes Leishmania genome plasticity through non-equivalent RAD9 and HUS1 functions.
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.