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Molecular and Cellular Biology, August 2005, p. 6617-6628, Vol. 25, No. 15
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.15.6617-6628.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

R2 Target-Primed Reverse Transcription: Ordered Cleavage and Polymerization Steps by Protein Subunits Asymmetrically Bound to the Target DNA

Shawn M. Christensen and Thomas H. Eickbush*

Department of Biology, University of Rochester, Rochester, New York 14627-0211

Received 29 March 2005/ Returned for modification 10 May 2005/ Accepted 16 May 2005

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.

* Corresponding author. Mailing address: Department of Biology, University of Rochester, Rochester, NY 14627. Phone: (585) 275-7247. Fax: (585) 275-2070. E-mail: eick{at}mail.rochester.edu.

Molecular and Cellular Biology, August 2005, p. 6617-6628, Vol. 25, No. 15
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.15.6617-6628.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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