Role of the Bombyx mori R2 element N-terminal domain in the target-primed reverse transcription (TPRT) reaction.
R2 is a site-specific non-long terminal repeat (non-LTR) retrotransposon encoding a single polypeptide with reverse transcriptase, DNA endonuclease and nucleic acid-binding domains. The current model of R2 retrotransposition involves an ordered series of cleavage and polymerization steps carried out by at least two R2 protein subunits, one bound upstream and one bound downstream of the integration site. The role in the retrotransposition reaction of two conserved DNA-binding motifs, a C2H2 zinc finger (ZF) and a Myb motif, located within the N-terminal domain of the protein are explored in this report. These motifs do not appear to play a role in RT or the ability of the protein to bind the R2 RNA transcript. Methylation and missing nucleoside interference-based DNA footprints using polypeptides to the N-terminal domain suggest the ZF and Myb motifs bind to regions -3 to -1 and +10 to +15 with reference to the insertion site. Mutations in these DNA sites or of the N-terminal protein domain blocked binding and the activity of the downstream subunit. Mutations of the protein domain also affected binding of the upstream subunit but not its function, suggesting the primary path to DNA target recognition by R2 involves both upstream and downstream subunits.
Christensen
Shawn M
SM
Department of Biology, University of Rochester, Rochester, NY 14627-0211, USA.
Bibillo
Arkadiusz
A
Eickbush
Thomas H
TH
D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D012831:Bombyx / Q000235:genetics; D004247:DNA / Q000378:metabolism; D018983:DNA Footprinting; D019476:Insect Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D020598:Proto-Oncogene Proteins c-myb / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018626:Retroelements; D048348:Reverse Transcription; D016335:Zinc Fingers
D019476:Insect Proteins; D010455:Peptides; D021122:Protein Subunits; D020598:Proto-Oncogene Proteins c-myb; D018626:Retroelements; D004247:DNA; C101001:R2 reverse transcriptase, Bombyx mori; D012194:RNA-Directed DNA Polymerase