The chromodomain of Tf1 integrase promotes binding to cDNA and mediates target site selection.
The long terminal repeat (LTR) retrotransposon Tf1 of Schizosaccharomyces pombe integrates specifically into the promoters of pol II-transcribed genes. Its integrase (IN) contains a C-terminal chromodomain related to the chromodomains that bind to the N-terminal tail of histone H3. Although we have been unable to detect an interaction between histone tails and the chromodomain of Tf1 IN, it is possible that the chromodomain plays a role in directing IN to its target sites. To test this idea, we generated transposons with single amino acid substitutions in highly conserved residues of the chromodomain and created a chromodomain-deleted mutant. The mutations, V1290A, Y1292A, W1305A, and CHDDelta, substantially reduced transposition activity in vivo. Blotting assays showed that there was little or no reduction in the levels of IN or cDNA. By measuring the homologous recombination between cDNA and the plasmid copy of Tf1, we found that two of the mutations did not reduce the import of cDNA into the nucleus, while another caused a 33% reduction. Chromatin immunoprecipitation assays revealed that CHDDelta caused an approximately threefold reduction in the binding of IN to the downstream LTR of the cDNA. These data indicate that the chromodomain contributed directly to integration. We therefore tested whether the chromodomain contributed to selecting insertion sites. Results of a target plasmid assay showed that the deletion of the chromodomain resulted in a drastic reduction in the preference for pol II promoters. Collectively, these data indicate that the chromodomain promotes binding of cDNA and plays a key role in efficient targeting.
Chatterjee
Atreyi Ghatak
AG
Section on Eukaryotic Transposable Elements, Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Leem
Young Eun
YE
Kelly
Felice D
FD
Levin
Henry L
HL
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D047369:Chromatin Immunoprecipitation; D018076:DNA, Complementary / Q000378:metabolism*; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D011995:Recombination, Genetic; D018626:Retroelements; D012568:Schizosaccharomyces / Q000235:genetics*; D016415:Sequence Alignment; D017384:Sequence Deletion
D018076:DNA, Complementary; D018626:Retroelements; D019426:Integrases