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...

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Bibliographic Details
Published in:Cellular and molecular life sciences : CMLS 2003-12, Vol.60 (12), p.2692-2701
Main Authors: García-Pérez, J L, González, C I, Thomas, M C, Olivares, M, López, M C
Format: Article
Language:English
Subjects:
Animals
Base Sequence
Binding Sites
Cloning, Molecular
DNA, Complementary - biosynthesis
Molecular Sequence Data
Proteins
Retroelements
RNA-Directed DNA Polymerase - genetics
RNA-Directed DNA Polymerase - metabolism
Trypanosoma cruzi
Trypanosoma cruzi - enzymology
Trypanosoma cruzi - genetics
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Summary: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.
ISSN:1420-682X
1420-9071
DOI:10.1007/s00018-003-3342-y