Genome-wide mapping of replication stress response factors reveals the management of polycistronic transcription, replication initiation, and responses to replication stress in Leishmania.

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.