Human ORF1p - DNA Interactions Characterized by Single Molecule DNA Stretching

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Retrotransposons are mobile genetic elements that possess the ability to amplify themselves in the genome via a process called retrotransposition. LINE-1 is a retrotransposon that comprises about 17% of the human genome, which is still active in most modern mammalian genomes. It is a significant source of interindividual genetic variations, defects and rearrangements. LINE-1 encodes two proteins: ORF1p and ORF2p, which are essential in retrotransposition. The role of ORF2p in retrotransposition as an endonuclease and as a reverse transcriptase has been demonstrated. However, the role of ORF1p is largely unknown, making the overall molecular mechanism of retrotransposition unclear. Studies on mouse ORF1p have revealed its nucleic acid chaperone activity, while human ORF1p (hORF1p) exhibits more complex nucleic acid interactions. Recent studies conducted in bulk solution conditions have shown that hORF1p preferentially binds to single-stranded DNA (ssDNA) and RNA relative to double-stranded DNA (dsDNA), but binds mismatched dsDNA with the same affinity as ssDNA, whereupon it stabilizes the mismatched duplex from dissociation. This property would enhance the production of productive primer-template interactions, a crucial step in the LINE-1 replication process. Here we develop a method to quantitatively characterize the mechanism of hORF1p-DNA interactions using single molecule techniques with optical tweezers. Because hORF1p binds strongly to both double- and single-stranded DNA, we first overstretch dsDNA, providing a lattice of ssDNA binding sites for hORF1p binding. We then flow in protein and incubate the stretched ssDNA for fixed times, followed by releasing the DNA and allowing it to anneal. We find that the amount of ssDNA bound by protein increases with incubation time over timescales of tens of minutes. These results suggest a very slow protein oligomerization process on ssDNA, which likely plays an important role in the mechanism of retrotransposition.