Abstract 1944 Importance of Domain 6a in R2Bm Binding Upstream of Target DNA

Long Interspersed Elements (LINEs) are retrotransposons found in almost all eukaryotic genomes. LINEs make up 17% of the human genome and although most are inactive, active LINEs can occasionally be responsible for diseases such as cancer, hemophilia, and muscular dystrophy. Studying LINEs is important for clinical implications, evolutionary tracking, and having a more in depth understanding of our genome (1). Understanding the mechanism of how LINEs integrate into the host genome could provide important insights into how other retrotransposons with similar mechanisms integrate and how these retrotransposons could be modified and used as gene delivery tools in the future. LINES insert into the host genome by two rounds of target primed DNA synthesis: Target Primed Reverse Transcription (TPRT) followed by target primed second-strand DNA synthesis (2). The biochemical aspects of LINE insertion have been most extensively worked out for Restriction Like Endonuclease (RLE)-LINEs, using the R2 element from Bombyx mori (R2Bm). R2 elements are site-specific and insert into the ribosomal genes of a wide range of animals. R2 elements encode a multifunctional protein with an N-terminal end containing a Zinc-Finger (ZF) and Myb motifs, a reverse transcriptase (RT) and a type IIs restriction-like endonuclease (RLE) towards the C-terminal end (2, 3). The element's protein binds to conserved regions of its own RNA. The 5' and 3' Protein Binding Motif (PBM) RNAs drive conformation changes of the R2 protein and of the integration complex. Biochemical studies, as well as a recent cryo-electron microscopy study, show that upstream binding is mediated via the ZnF, Myb and domain 6a (4). In this study, we use in vitro reactions with mutant R2 proteins to extend these earlier studies and focus on the importance of domain 6a in target recognition in the presence of 3' PBM RNA. Our data indicate that the protein DNA interaction surface mediated by domain 6a is able to withstand the loss of several of the phosphate contacts and still function. NSF.