Characterizing complex nucleic acid interactions of LINE1 ORF1p by single molecule force spectroscopy

Abstract

The L1 retrotransposon is the dominant transposable element in mammalian genomes. L1 comprises at least 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, which are required for retrotransposition. During retrotransposition, ORF2p functions as the reverse transcriptase and the endonuclease. ORF1p is a nucleic acid chaperone that binds nucleic acids with high affinity. However, to date, a detailed mechanistic understanding of ORF1p function in L1 retrotransposition is lacking. The single molecule DNA stretching methods described here have been extensively used to understand ORF1p’s complex nucleic acid binding properties. By correlating these properties to ORF1p’s ability to support L1 retrotransposition in in vivo cell-culture based assays, these studies have significantly contributed to advance the understanding of ORF1p function. Although described in the context of ORF1p, these methods provide a general mechanism to study complex protein-DNA interactions.