Conditional genome engineering reveals canonical and divergent roles for the Hus1 component of the 9-1-1 complex in the maintenance of the plastic genome of Leishmania.

Leishmania species are protozoan parasites whose remarkably plastic genome limits the establishment of effective genetic manipulation and leishmaniasis treatment. The strategies used by Leishmania to maintain its genome while allowing variability are not fully understood. Here, we used DiCre-mediated conditional gene deletion to show that HUS1, a component of the 9-1-1 (RAD9-RAD1-HUS1) complex, is essential and is required for a G2/M checkpoint. By analyzing genome-wide instability in HUS1 ablated cells, HUS1 is shown to have a conserved role, by which it preserves genome stability and also a divergent role, by which it promotes genome variability. These roles of HUS1 are related to distinct patterns of formation and resolution of single-stranded DNA and γH2A, throughout the cell cycle. Our findings suggest that Leishmania 9-1-1 subunits have evolved to co-opt canonical genomic maintenance and genomic variation functions. Hence, this study reveals a pivotal function of HUS1 in balancing genome stability and transmission in Leishmania. These findings may be relevant to understanding the evolution of genome maintenance and plasticity in other pathogens and eukaryotes.

Unpicking the Roles of DNA Damage Protein Kinases in Trypanosomatids.

To preserve genome integrity when faced with DNA lesions, cells activate and coordinate a multitude of DNA repair pathways to ensure timely error correction or tolerance, collectively called the DNA damage response (DDR). These interconnecting damage response pathways are molecular signal relays, with protein kinases (PKs) at the pinnacle. Focused efforts in model eukaryotes have revealed intricate aspects of DNA repair PK function, including how they direct DDR pathways and how repair reactions connect to wider cellular processes, including DNA replication and transcription. The Kinetoplastidae, including many parasites like Trypanosoma spp. and Leishmania spp. (causative agents of debilitating, neglected tropical infections), exhibit peculiarities in several core biological processes, including the predominance of multigenic transcription and the streamlining or repurposing of DNA repair pathways, such as the loss of non-homologous end joining and novel operation of nucleotide excision repair (NER). Very recent studies have implicated ATR and ATM kinases in the DDR of kinetoplastid parasites, whereas DNA-dependent protein kinase (DNA-PKcs) displays uncertain conservation, questioning what functions it fulfills. The wide range of genetic manipulation approaches in these organisms presents an opportunity to investigate DNA repair kinase roles in kinetoplastids and to ask if further kinases are involved. Furthermore, the availability of kinase inhibitory compounds, targeting numerous eukaryotic PKs, could allow us to test the suitability of DNA repair PKs as novel chemotherapeutic targets. Here, we will review recent advances in the study of trypanosomatid DNA repair kinases.

A DiCre recombinase-based system for inducible expression in Leishmania major.

Here we present the establishment of an inducible system based on the dimerizable Cre recombinase (DiCre) for controlled gene expression in the protozoan parasite Leishmania. Rapamycin-induced DiCre activation promoted efficient flipping and expression of gene products in a time and dose-dependent manner. The DiCre flipping activity induced the expression of target genes from both integrated and episomal contexts broadening the applicability of the system. We validated the system by inducing the expression of both full length and truncated forms of the checkpoint protein Rad9, which revealed that the highly divergent C-terminal domain of Rad9 is necessary for proper subcellular localization. Thus, by establishing the DiCre-based inducible system we have created and validated a robust new tool for assessing gene function in Leishmania.