Endogenous overexpression of an active phosphorylated form of DNA polymerase β under oxidative stress in Trypanosoma cruzi

Abstract

Trypanosoma cruzi is exposed during its life to exogenous and endogenous oxidative stress, leading to damage of several macromolecules such as DNA. There are many DNA repair pathways in the nucleus and mitochondria (kinetoplast), where specific protein complexes detect and eliminate damage to DNA. One group of these proteins is the DNA polymerases. In particular, Tc DNA polymerase β participates in kinetoplast DNA replication and repair. However, the mechanisms which control its expression under oxidative stress are still unknown. Here we describe the effect of oxidative stress on the expression and function of Tc DNA polymerase β To this end parasite cells (epimastigotes and trypomastigotes) were exposed to peroxide during short periods of time. Tc DNA polymerase β which was associated physically with kinetoplast DNA, showed increased protein levels in response to peroxide damage in both parasite forms analyzed. Two forms of DNA polymerase β were identified and overexpressed after peroxide treatment. One of them was phosphorylated and active in DNA synthesis after renaturation on polyacrylamide electrophoresis gel. This phosphorylated form showed 3-4-fold increase in both parasite forms. Our findings indicate that these increments in protein levels are not under transcriptional control because the level of Tc DNA polymerase β mRNA is maintained or slightly decreased during the exposure to oxidative stress. We propose a mechanism where a DNA repair pathway activates a cascade leading to the increment of expression and phosphorylation of Tc DNA polymerase β in response to oxidative damage, which is discussed in the context of what is known in other trypanosomes which lack transcriptional control.

Analysis of DNA Polymerases Reveals Specific Genes Expansion in Leishmania and Trypanosoma spp.

“…In trypanosomatids, Pol β is perhaps one of the most studied TLS polymerases. While only one POLB gene has been described for Leishmania spp (Alonso et al, 2006 ; Khan et al, 2019 ), mainly located at the nucleus, in T. cruzi (de Oliveira Lopes et al, 2008 ; Schamber-reis et al, 2012 ; Rojas et al, 2018 ) and T. brucei (Saxowsky et al, 2003 ), the enzyme is mainly located in mitochondria. Interestingly, in T. brucei , two Pol β variants have been described: Pol β and Pol β-PAK (an Nt domain rich in proline-alanine-lysine, only found in these protozoans).…”

“…Experimental data remains to be carried out to confirm if they are real homologs. In T. cruzi has been shown that Pol β and Pol β-PAK-like have dRP lyase activity, and moreover Pol β-PAK-like is able to bypass 7,8-dihydro-8-oxoguanine (8-oxoG), strongly suggesting specialized roles in BER and TLS for kDNA oxidative protection and maintenance (de Oliveira Lopes et al, 2008 ; Rojas et al, 2018 ).…”

Dual and Opposite Roles of Reactive Oxygen Species (ROS) in Chagas Disease: Beneficial on the Pathogen and Harmful on the Host

“…The high form is heavily phosphorylated (H), while the low form is unphosphorylated (L). Interestingly, the high form is more active in in vitro DNA synthesis [ 96 ]. The levels of the phosphorylated active form of the enzyme are higher in H 2 O 2 -treated cells compared with untreated cells, suggesting that this activity is involved in repair of the kinetoplast DNA and perhaps is also responsible of the kinetoplast DNA replication [ 96 ].…”

“…Interestingly, the high form is more active in in vitro DNA synthesis [ 96 ]. The levels of the phosphorylated active form of the enzyme are higher in H 2 O 2 -treated cells compared with untreated cells, suggesting that this activity is involved in repair of the kinetoplast DNA and perhaps is also responsible of the kinetoplast DNA replication [ 96 ]. A mechanistic model to explain the overexpression of Tcpol β is outlined in Figure 3 and it can be observed the different protein families involved in the posttranscriptional expression of the enzyme.…”

“…A mechanistic model to explain the overexpression of Tcpol β is outlined in Figure 3 and it can be observed the different protein families involved in the posttranscriptional expression of the enzyme. Tcpol β can be crosslinked to kinetoplast DNA, but no to nuclear DNA, indicating that the enzyme is always in contact with kinetoplast DNA [ 96 ]. We believe that several protein kinases are involved in the phosphorylation of Tcpol β and those kinases are involved in the signal transduction pathway triggered by the H 2 O 2 treatment.…”