Characterization of uracil-DNA glycosylase activity from Trypanosoma cruzi and its stimulation by AP endonuclease

Abstract

The intracellular pathogen Trypanosoma cruzi is the etiological agent of Chagas' disease. We have isolated a full-length cDNA encoding uracil-DNA glycosylase (UDGase), a key enzyme involved in DNA repair, from this organism. The deduced protein sequence is highly conserved at the C-terminus of the molecule and shares key residues involved in binding or catalysis with most of the UDGases described so far, while the N-terminal part is highly variable. The gene is single copy and is located on a chromosome of approximately 1.9 Mb. A His-tagged recombinant protein was overexpressed, purified and used to raise polyclonal antibodies. Western blot analysis revealed the existence of a single UDGase species in parasite extracts. Using a specific ethidium bromide fluorescence assay, recombinant T.cruzi UDGase was shown to specifically excise uracil from DNA. The addition of both Leishmania major AP endonuclease and exonuclease III, the major AP endonuclease from Escherichia coli, produces stimulation of UDGase activity. This activation is specific for AP endonuclease and suggests functional communication between the two enzymes.

Figure 1

Structure-based alignment of UDGase sequences (Swiss-Prot accession nos in parentheses): human (P13051), H.influenzae (P43731), equine herpes virus type I (P28866), S.cerevisiae (P12887) and T.cruzi. The GenBank accession no. for the T.cruzi UDGase cDNA sequence is AF152347. The secondary structure motifs identified in the crystal structure of human UDGase are indicated above the sequence alignment (26,35). The consensus sequence is also shown.

Figure 2

SDS–PAGE of purified UDGase. Lane 1, positions of molecular mass standards; lane 2, soluble BL21(DE3)/pETTcung cell extracts without IPTG induction; lane 3, soluble BL21(DE3)/ pETTcung extracts after 2 h induction with IPTG; lanes 4 and 5, fractions of purified UDGase after affinity column elution.

Figure 3

Western blot analysis of T.cruzi UDGase. Lane 1, purified recombinant UDGase; lane 2, sonicated soluble extracts of the T.cruzi Y strain. Samples were made to react with the polyclonal antibodies and detection was performed as described in Materials and Methods.

Figure 4

Assay of T.cruzi UDGase. Closed squares correspond to ethidium bromide fluorescence of cccDNA after heating the samples at 95°C for 8 min. Open squares correspond to ethidium bromide fluorescence after treating the samples with 2 vol DMSO. The data at time 0 were normalized to 100%. (A) Assay using uracil-containing cccDNA. (B) Assay using control cccDNA.

Figure 5

Enhancement of UDGase activity by LmAP. Decrease in fluorescence observed in the presence of different concentrations of LmAP. Open squares, UDGase; closed squares, UDGase + 2LmAP; open triangles, UDGase + 10 molar excess LmAP; closed triangles, UDGase + 50 molar excess LmAP; open circles, UDGase + 100 molar excess LmAP; closed circles, LmAP.

Figure 6

Enhancement of UDGase activity by exonuclease III. Decrease in fluorescence observed in the presence of different concentrations of exonuclease III. Open squares, UDGase; closed squares, UDGase + 40 U; triangles, UDGase +80 U; open circles, UDGase +175 U; closed circles, LmAP.