Expression, purification, and biochemical characterization of recombinant DNA polymerase beta of the Trypanosoma cruzi TcI lineage: requirement of additional factors and detection of phosphorylation of the native form

Abstract

Chagas disease, caused by the protozoan Trypanosoma cruzi, is a major parasitic disease that affects millions of people in America. However, despite the high impact of this disease on human health, no effective and safe treatment has been found that eliminates the infecting parasite from human patients. Among the possible chemotherapeutic targets that could be considered for study in T. cruzi are the DNA polymerases, in particular DNA polymerase beta (polß), which previous studies have shown to be involved in kinetoplast DNA replication and repair. In this paper, we describe the expression, purification, and biochemical characterization of the Miranda clone polß, corresponding to lineage T. cruzi I (TcI). The recombinant enzyme purified to homogeneity displayed specific activity in the range described for a highly purified mammalian polß. However, the trypanosome enzyme exhibited important differences in biochemical properties compared to the mammalian enzymes, specifically an almost absolute dependency on KCl, high sensitivity to N-ethylmaleimide (NEM), and low sensitivity to ddTTP. Immuno-affinity purification of T. cruzi polymerase beta (Tcpolß) from epimastigote extracts showed that the native enzyme was phosphorylated. In addition, it was demonstrated that Tcpolß interacts with some proteins in a group of about 15 proteins which are required to repair 1-6 bases of gaps of a double strand damaged DNA. It is possible that these proteins form part of a DNA repair complex, analogous to that described in mammals and some trypanosomatids.

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

“…In the precipitated material, the slower migrating polypeptide (H) is phosphorylated in trypomastigote cells, since it can react with antibodies against phosphoaminoacids ( Fig 2C ). These observations agree with previous results in epimastigotes [ 27 ]. We did not detect a change in the mobility of the slower migrating band upon treatment with N- or O- glycosidases, suggesting the enzyme does not contain sugar moieties.…”

“…We have previously purified and characterized the native and recombinant DNA polymerase β from T . cruzi epimastigotes and generated polyclonal antibodies [ 26 , 27 ]. To gain insights into the role of DNA polymerase β in T .…”

T. cruzi DNA polymerase beta (Tcpolβ) is phosphorylated in vitro by CK1, CK2 and TcAUK1 leading to the potentiation of its DNA synthesis activity

“…Tcpolβ has multiple predicted phosphorylation sites for several protein kinases, however, it is unknown whether any of those can phosphorylate the enzyme and whether or not the event has any consequence on the activity of the enzyme [17,19]. In an earlier work [17], predicted phosphorylation sites for CK1, CK2, PKA and PKC were found in Tcpolβ, however, those sites are not in similar positions as those found in the mammalian homologue. Using bioinformatic tools, set at high stringency, we were able to detect phosphorylation sites for casein kinase 1 and 2 (CK1, CK2) and aurora kinase (AUK).…”

“…The gene encoding DNA polymerase β from T. cruzi (Tcpolβ) has been cloned and the recombinant protein was expressed and studied [15][16][17]. Tcpolβ locates to the kinetoplast of the cell and it can be crosslinked to the DNA of that organelle, however, it cannot be crosslinked to nuclear DNA [18,19].…”

“…It possesses intrinsic 5 -deoxyribose phosphate (dRP) lyase activity, which in part contributes to the kDNA repair of oxidative lesions through the BER repair system [16,18]. Tcpolβ can repair 1-6 bases of gaps of a double strand damaged DNA in the presence of additional T. cruzi proteins in in vitro assays, which could indicate that in vivo is part of a multiprotein complex [17]. Also, when parasites are exposed to high doses of hydrogen peroxide, respond overexpressing Tcpolβ.…”