Completion of RLE LINE Integration Involves an Open "4-Way" Branched DNA Intermediate

Abstract

Long Interspersed Elements (LINEs), also known as non-LTR retrotransposons, encode a multifunctional protein that reverse transcribes its mRNA into DNA at the site of insertion by target primed reverse transcription. The R2 Long Interspersed Elements (LINEs) specifically integrate in the 28S rRNA genes by a series of DNA binding, DNA cleavage, and DNA synthesis reactions. While the first half of the integration reaction, TPRT, is well understood, the second half of the integration reaction, second-strand DNA cleavage and second-strand DNA synthesis are much less well understood. A hitherto unknown and unexplored branched integration intermediate, an open ‘4-way’ DNA junction which is thoguht to arise by template jumping, was recognized by the element protein and cleaved in a Holliday junction resolvase-like reaction. Cleavage of the branched integration intermediate resulted in a natural primer-template pairing used for second- strand DNA synthesis. In addition, the structure of the branched integration intermediate itself was explored by probing with DNase I footprint and was found to be highly structured. R2 protein binding to the junction was explored by a combination of DNA cleavage assays and DNA footprint studies. The protein appears to bind in a sequence specific manner to the downstream sequence of branched integration intermediates, but less so for the upstream sequence where structure appears to be more important. A new model for RLE LINE integration is presented.