Characterizing Complex Nucleic Acid Interactions of LINE1 ORF1p by Single-Molecule Force Spectroscopy

The LINE-1 (L1) retrotransposon is the dominant transposable element in mammalian genomes and comprises ~20% of the human genome. While most L1 regions are inactive, a few still retain the ability to retrotranspose. L1 encodes two proteins, ORF1p and ORF2p, that are required for retrotransposition. ORF2p functions as the reverse transcriptase and endonuclease. ORF1p is a nucleic acid (NA) chaperone that binds single-stranded (ss) NA with high affinity. However, to date, a detailed mechanistic understanding of ORF1p function in L1 retrotransposition is lacking. The methods described here utilize single molecule DNA-stretching techniques, which have been extensively used to dissect ORF1p's complex binding interactions with both single-stranded and double-stranded DNAs. By correlating these properties with ORF1p's ability to support L1 retrotransposition in in vivo cell-culture-based assays, these studies have significantly advanced our understanding of ORF1p function. Although described in the context of ORF1p, these methods provide a general procedure for studying complex protein-DNA interactions.