Footprint of the retrotransposon R2Bm protein on its target site before and after cleavage.
R2 elements are non-long terminal repeat (non-LTR) retrotransposons that specifically integrate into the 28 S rRNA genes of their host. These elements encode a single open reading frame with a genome-specific endonuclease and a reverse transcriptase that uses the cleaved chromosomal target site to prime reverse transcription. Cleavage of the DNA strand that is used to prime reverse transcription is an efficient process that occurs in the presence or absence of RNA. Cleavage of the second DNA strand is much less efficient and requires RNA. Reverse transcription occurs before second strand cleavage and only if the RNA bound to the protein contains the 3' untranslated region of the R2 element. Thus a complex series of protein interactions with the DNA and conformational changes in the protein are likely to occur during this retrotransposition reaction. Here, we conduct electrophoretic mobility-shift assays and DNase I footprint studies on the binding of the R2 protein to the DNA target in the presence and absence of RNA both before and after first strand cleavage. While the total expanse of the protein footprint on the DNA eventually covers five helical turns, before cleavage the footprint only extends from 17 bp to 40 bp upstream of the cleavage site. This footprint is the same in the presence and absence of RNA. We hypothesize that the active site of the endonuclease domain is analogous to type IIS restriction enzymes in that it is located on a flexible domain that is not tightly bound to the cleavage site. After first strand cleavage the protein footprint extends beyond the cleavage site. We suggest that this increased protection after cleavage is the RT domain that is positioned over the free DNA end to begin reverse transcription on the nicked DNA substrate.
Christensen
Shawn
S
Department of Biology, University of Rochester, Hutchinson Hall 334, Rochester, NY 14627-0211, USA.
Eickbush
Thomas H
TH
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D012831:Bombyx; D004275:DNA, Ribosomal / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004720:Endonucleases / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D023302:Protein Footprinting; D017434:Protein Structure, Tertiary; D012339:RNA, Ribosomal, 28S; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D018626:Retroelements
D004275:DNA, Ribosomal; D004268:DNA-Binding Proteins; D012339:RNA, Ribosomal, 28S; D018626:Retroelements; D012194:RNA-Directed DNA Polymerase; D004720:Endonucleases; D003850:Deoxyribonuclease I