High Frequency cDNA Recombination of the Saccharomyces Retrotransposon Ty5: The LTR Mediates Formation of Tandem Elements

Abstract

Retroelement cDNA can integrate into the genome using the element-encoded integrase, or it can recombine with preexisting elements using the recombination system of the host. Recombination is a particularly important pathway for the yeast retrotransposon Ty5 and accounts for ∼30% of the putative transposition events when a homologous substrate is carried on a plasmid and ∼7% when the substrate is located at the chromosomal URA3 locus. Characterization of recombinants revealed that they are either simple replacements of the marker gene or tandem elements. Using an assay system in which the donor element and recombination substrates are separated, we found that the long terminal repeats (LTRs) are critical for tandem element formation. LTR-containing substrates generate tandem elements at frequencies more than 10-fold higher than similarly sized internal Ty5 sequences. Internal sequences, however, facilitate tandem element formation when associated with an LTR, and there is a linear relationship between frequencies of tandem element formation and the length of LTR-containing substrates. We propose that recombination is initiated between the LTRs of the cDNA and substrate and that internal sequences promote tandem element formation by facilitating sequence alignment. Because of its location in subtelomeric regions, recombinational amplification of Ty5 may contribute to the organization of chromosome ends.

Phosphorylation Regulates Integration of the Yeast Ty5 Retrotransposon into Heterochromatin

“…The GST fusion constructs were generated in pEG(KG) (Mitchell et al, 1993) by inserting a DNA fragment obtained either by PCR amplification and cloning or by annealing two complementary oligonucleotides. PCR-based mutagenesis (Ausubel et al, 1987) was used to introduce the TD mutations into a full-length Ty5 element on pNK254 (Ke and Voytas, 1997). This was accomplished by PCR amplifying pNK254 with two pairs of primers: (1) a forward primer and mutagenic primer, and (2) a reverse primer and a second, complementary mutagenic primer.…”

Identification of an integrase-independent pathway of retrotransposition

“…HIV-1 can retain residual insertion activity when IN function is disrupted with mutations or potent inhibitors (6)(7)(8)(9)(10)(11). In addition, INindependent insertion of MLV in human embryonic kidney 293T cells and of LTR retrotransposons in S. cerevisiae indicates that an INindependent pathway is a secondary and common strategy of insertion for retroviruses and retrotransposons (13,(40)(41)(42). Although host DNA repair machinery has been implicated in IN-independent insertion of HIV-1, little is understood about the mechanism (11).…”

“…Tandem repeats of LTR retrotransposons are observed in a wide range of eukaryotic species, including S. pombe, S. cerevisiae, several bivalve species, and a number of plant species (31,(40)(41)(42)(56)(57)(58)(59)(60)(61). A computational pipeline that specifically detects tandem elements showed in Drosophila melanogaster sampled across five continents that most LTR retrotransposons form tandem repeats (62).…”

Light and shadow on the mechanisms of integration site selection in yeast Ty retrotransposon families

“…Regarding Ty3, the transcription of the two endogenous copies is repressed by competing Pol III transcription of the adjacent tDNAs, resulting in extremely low level of expression (Bilanchone et al, 1993;Hull et al, 1994;Kinsey and Sandmeyer, 1991). S. cerevisiae does not have a functional Ty5 element but the introduction of an active Ty5 element from S. paradoxus revealed that the heterochromatin targeted by the element repressed the transcription of de novo Ty5 insertions (Ke et al, 1997).…”

“…Yet, the expression of both Ty3 and Ty5 is strongly stimulated in haploid cells exposed to pheromones, allowing the transposition of active copies in particular conditions (Bilanchone et al, 1993;Ke et al, 1997;Kinsey and Sandmeyer, 1995). Switching between repression and activation of Ty expression undoubtedly may help minimize the adverse effects of their propagation while maintaining a relatively low copy number in the cells.…”