Resolving the polycistronic aftermath: Essential role of topoisomerase IA in preventing R-loops in Leishmania

Kinetoplastid parasites are "living bridges" in the evolution from prokaryotes to higher eukaryotes. The near-intronless genome of the kinetoplastid Leishmania exhibits polycistronic transcription which can facilitate R-loop formation. Therefore, to prevent such DNA-RNA hybrids, Leishmania has retained prokaryotic-like DNA Topoisomerase IA (LdTOPIA) in the course of evolution. LdTOPIA is an essential enzyme that is expressed ubiquitously and is adapted for the compartmentalized eukaryotic form in harboring functional bipartite nuclear localization signals. Although exhibiting greater homology to mycobacterial TOPIA, LdTOPIA could functionally complement the growth lethality of Escherichia coli TOPIA null GyrB ts strain at non-permissive temperatures. Purified LdTOPIA exhibits Mg²⁺-dependent relaxation of only negatively supercoiled DNA and preference towards single-stranded DNA substrates. LdTOPIA prevents nuclear R-loops as conditional LdTOPIA downregulated parasites exhibit R-loop formation and thereby parasite killing. The clinically used tricyclic antidepressant, norclomipramine could specifically inhibit LdTOPIA and lead to R-loop formation and parasite elimination. This comprehensive study therefore paves an avenue for drug repurposing against Leishmania.