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Restriction-like endonuclease (RLE) bearing non-LTR retrotransposons are site-specific elements that integrate into the genome through target primed reverse transcription (TPRT). RLE-bearing elements have been used as a model system for investigating non-LTR retrotransposon integration. R2 elements target a specific site in the 28S rDNA gene. We previously demonstrated that the two major sub-classes of R2 (R2-A and R2-D) target the R2 insertion site in an opposing manner with regard to the pairing of known DNA binding domains and bound sequences-indicating that the A- and D-clades represent independently derived modes of targeting that site. Elements have been discovered that group phylogenetically with R2 but do not target the canonical R2 site. Here we extend our earlier studies to show that a separate R2-A clade element, which targets a site other than the canonical R2 site, does so by using the N-terminal zinc fingers and Myb motifs. We further extend our targeting studies beyond R2 clade elements by investigating the ability of the N-terminal zinc fingers from the nematode NeSL-1 element to target its integration site. Our data are consistent with the use of an N-terminal DNA binding domain as one of the major targeting determinants used by RLE-bearing non-LTR retrotransposons to secure a protein subunit near the insertion site. This N-terminal DNA binding domain can undergo modifications, allowing the element to target novel sites. The binding orientation of the N-terminal domain relative to the insertion site is quite variable.

Restriction-like endonuclease (RLE) bearing non-LTR retrotransposons are site-specific elements that integrate into the genome through a target primed reverse transcription mechanism (TPRT). R2 elements have been used as a model system for investigating non-LTR retrotransposon integration. We previously demonstrated that R2 retrotransposons require two subunits of the element-encoded multifunctional protein to integrate-one subunit bound upstream of the insertion site and one bound downstream. R2 elements have been phylogenetically categorized into four clades: R2-A, B, C and D, that diverged from a common ancestor more than 850 million years ago. All R2 elements target the same sequence within 28S rDNA. The amino-terminal domain of R2Bm, an R2-D clade element, contains a single zinc finger and a Myb motif that are responsible for binding R2 protein downstream of the insertion site. Target site recognition is of interest as it is the first step in the integration reaction and may help elucidate evolutionary history and integration mechanism. The amino-terminal domain of R2-A clade members contains three zinc fingers and a Myb motif. We show here that R2Lp, an R2-A clade member, uses its amino-terminal DNA binding motifs to bind upstream of the insertion site. Because the R2-A and R2-D clade elements recognize 28S rDNA differently, we conclude the A- and D-clades represent independent targeting events to the 28S site. Our results also indicate a certain plasticity of insertional mechanics exists between the two clades.

R2 elements are non-long terminal repeat retrotransposons that specifically insert into 28S rRNA genes of many animal groups. These elements encode a single protein with reverse transcriptase and endonuclease activities as well as specific DNA and RNA binding properties. In this report, gel shift experiments were conducted to investigate the stoichiometry of the DNA, RNA, and protein components of the integration reaction. The enzymatic functions associated with each of the protein complexes were also determined, and DNase I digests were used to footprint the protein onto the target DNA. Additionally, a short polypeptide containing the N-terminal putative DNA-binding motifs was footprinted on the DNA target site. These combined findings revealed that one protein subunit binds the R2 RNA template and the DNA 10 to 40 bp upstream of the insertion site. This subunit cleaves the first DNA strand and uses that cleavage to prime reverse transcription of the R2 RNA transcript. Another protein subunit(s) uses the N-terminal DNA binding motifs to bind to the 18 bp of target DNA downstream of the insertion site and is responsible for cleavage of the second DNA strand. A complete model for the R2 integration reaction is presented, which with minor modifications is adaptable to other non-LTR retrotransposons.