Characterization of reverse transcriptase activity of the L1Tc retroelement from Trypanosoma cruzi.
The recombinant protein RTL1Tc, encoded by the non-LTR (long terminal repeat) retrotransposon L1Tc from Trypanosoma cruzi, has been shown to have reverse transcriptase (RT) activity using poly(rA)/oligo(dT) and poly(rC)/oligo(dG) homopolymers as template/primers. The optimal RT activity was detected at a concentration of 5 mM Mg2+, pH 8 and between 28 and 37% degrees C. Site-directed mutagenesis in the RT catalytic site proved that substitution of aspartic acid 313 for isoleucine (RT D313IL1Tc) practically abolishes the RT activity of the RTL1Tc protein. RT-polymerase chain reaction assays revealed that the RTL1Tc protein has the ability to use both homologous and heterologous RNA templates. Also, it is shown that the RTL1Tc protein is capable of synthesizing complementary DNA molecules by consecutive switching of the oligo molecule, which the protein uses as a template. This template switching may be involved in the retroelement integration process.
García-Pérez
J L
JL
Departamento de Biología Molecular, Instituto de Parasitología y Biomedicina López Neyra, Calle Ventanilla, 11, 18001 Granada, Spain.
González
C I
CI
Thomas
M C
MC
Olivares
M
M
López
M C
MC
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis; D008969:Molecular Sequence Data; D012194:RNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism; D018626:Retroelements; D014349:Trypanosoma cruzi / Q000201:enzymology / Q000235:genetics*
D018076:DNA, Complementary; D018626:Retroelements; D012194:RNA-Directed DNA Polymerase