Untersuchungen zur positionsspezifischen Integration des LTR-Retrotransposons DGLT-A im Genom von Dictyostelium discoideum

Kling, Eva GND

Transposable elements (TE) are able to amplify as selfish DNA elements in genomes. Their intrinsic integration can cause insertional mutagenesis of protein-coding genes and challenge their hosts fitness. To avoid compromising the genome integrity some TEs developed mechanisms for site specific integration. This study describes the molecular mechanism of position specific integration by the LTR retrotransposon DGLT A in the social amoeba Dictyostelium discoideum. In the gene dense haploid genome of D. discoideum DGLT A integrates position specifically 15 bp upstream of tRNA genes. DGLT A uses contacts with the preinitiation complex of RNA polymerase III, which is composed of the complexes TFIIIB and TFIIIC, to identify integration sites upstream of tRNA genes. Yeast two hybrid screening and in vitro pulldown experiments determined that DGLT A uses two retrotransposon encoded proteins to interact with the TFIIIC complex: ribonuclease H (RNH) and integrase (IN). Both proteins interact with TFIIIC subunit Tfc4. Tfc4 contains 12 tetratricopeptide repeats (TPRs) of which TPR1 10 are arranged in a central TPR array. We discovered that DGLT A RNH and IN interact with an interface that covers TPR 7 and 8. A major function of this interface is to connect TFIIIC subcomplexes A and B and to facilitate TFIIIB assembly in the RNA polymerase III transcription complex. Thus, we hypothesize that the DGLT A intasome gets access to genomic DNA for integration during tRNA gene transcription by competing with B for binding at theTPR 7/8 surface in A. Results from this study suggest that the distantly related amoebozoan D. discoideum LTR retrotransposon DGLT A and the yeast Saccharomyces cerevisiae element Ty3 evolved targeted integration upstream of tRNA genes by convergent evolution. Both retrotransposons solved the problem of finding safe integration sites in compact genomes by targeting to regions close to tRNA genes, but they use different molecular mechanisms to identify their target site 15 bp upstream of tRNA genes. Whereas Ty3 encoded integrase interacts with the TBP/Brf1 heterodimer of TFIIIB suggesting competition of Ty3 during the initiation of POLIII transcription, DGLT A RNH and IN presumably compete with A/B interaction during transcription elongation.



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