R2 target-primed reverse transcription: ordered cleavage and polymerization steps by protein subunits asymmetrically bound to the target DNA.
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.
Christensen
Shawn M
SM
Department of Biology, University of Rochester, Rochester, NY 14627, USA.
Eickbush
Thomas H
TH
D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000201:enzymology / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism; D006868:Hydrolysis; D019476:Insect Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism*; D012313:RNA / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D018626:Retroelements / Q000235:genetics / Q000502:physiology*; D020079:Terminal Repeat Sequences / Q000235:genetics / Q000502:physiology*
D004268:DNA-Binding Proteins; D019476:Insect Proteins; D021122:Protein Subunits; D018626:Retroelements; D012313:RNA; C101001:R2 reverse transcriptase, Bombyx mori; D012194:RNA-Directed DNA Polymerase; D004720:Endonucleases