DNA topoisomerases are
enzymes that modulate
DNA topology. Among them, topoisomerase 3α is engaged in genomic
maintenance acting in
DNA replication termination,
sister chromatid separation, and
dissolution of
recombination intermediates. To evaluate the
role of this
enzyme in
Trypanosoma cruzi, the etiologic agent of
Chagas disease, a topoisomerase 3α knockout
parasite (TcTopo3α KO) was generated, and the
parasite growth, as well as its response to several
DNA damage agents, were evaluated. There was no
growth alteration caused by the TcTopo3α knockout in epimastigote forms, but a higher dormancy rate was observed. TcTopo3α KO trypomastigote forms displayed reduced invasion rates in LLC-MK2
cells when compared with the wild-type lineage. Amastigote proliferation was also compromised in the TcTopo3α KO, and a higher number of dormant
cells was observed. Additionally, TcTopo3α KO epimastigotes were not able to recover
cell growth after
gamma radiation exposure, suggesting the involvement of topoisomerase 3α in
homologous recombination. These
parasites were also sensitive to
drugs that generate replication stress, such as
cisplatin (Cis),
hydroxyurea (HU), and
methyl methanesulfonate (MMS). In response to HU and Cis
treatments, TcTopo3α KO
parasites showed a slower
cell growth and was not able to efficiently repair the
DNA damage induced by these genotoxic agents. The
cell growth phenotype observed after MMS
treatment was
similar to that observed after
gamma radiation, although there were fewer dormant
cells after MMS exposure. TcTopo3α KO
parasites showed a
population with sub-G1
DNA content and strong γH2A signal 48 h after MMS
treatment. So, it is possible that
DNA-damaged
cell proliferation due to the absence of TcTopo3α leads to
cell death.
Whole genome sequencing of MMS-treated
parasites showed a significant reduction in the content of the
multigene families DFG-1 and RHS, and also a possible
erosion of the sub-telomeric region from
chromosome 22, relative to non-treated knockout
parasites.
Southern blot experiments suggest
telomere shortening, which could indicate
genomic instability in TcTopo3α KO
cells owing to MMS
treatment. Thus, topoisomerase 3α is important for
homologous recombination repair and replication stress in T. cruzi, even though all the pathways in which this
enzyme participates during the replication stress response remains elusive.