title: Endonuclease domain of non-LTR retrotransposons:  loss-of-function mutants and modeling of the R2Bm  endonuclease.
abstract: Non-LTR retrotransposons are an important class of mobile elements that insert into host DNA by target-primed reverse transcription (TPRT). Non-LTR retrotransposons must bind to their mRNA, recognize and cleave their target DNA, and perform TPRT at the site of DNA cleavage. As DNA binding and cleavage are such central parts of the integration reaction, a better understanding of the endonuclease encoded by non-LTR retrotransposons is needed. This paper explores the R2 endonuclease domain from Bombyx mori using in vitro studies and in silico modeling. Mutations in conserved sequences located across the putative PD-(D/E)XK endonuclease domain reduced DNA cleavage, DNA binding and TPRT. A mutation at the beginning of the first α-helix of the modeled endonuclease obliterated DNA cleavage and greatly reduced DNA binding. It also reduced TPRT when tested on pre-cleaved DNA substrates. The catalytic K was located to a non-canonical position within the second α-helix. A mutation located after the fourth β-strand reduced DNA binding and cleavage. The motifs that showed impaired activity form an extensive basic region. The R2 biochemical and structural data are compared and contrasted with that of two other well characterized PD-(D/E)XK endonucleases, restriction endonucleases and archaeal Holliday junction resolvases.
authors.forename: Aruna
authors.initials: A
authors.lastname: Govindaraju
authors.forename: Jeremy D
authors.initials: JD
authors.lastname: Cortez
authors.forename: Brad
authors.initials: B
authors.lastname: Reveal
authors.forename: Shawn M
authors.initials: SM
authors.lastname: Christensen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D012831:Bombyx / Q000201:enzymology; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism; D053837:DNA Cleavage; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D017433:Protein Structure, Secondary; D018626:Retroelements; D048348:Reverse Transcription; D016415:Sequence Alignment
chemical_list: D018626:Retroelements; D004247:DNA; D004706:Endodeoxyribonucleases