Single Molecule Binding Dynamics of Line-1 ORF1P to SSDNA

Abstract

Long interspersed nuclear element 1 (LINE-1) is an intragenomic parasitic element that makes up ∼ 20% of the human genome. It amplifies in the host genome by copying its RNA transcript into genomic DNA through a process called retrotransposition. L1 encodes two proteins, ORF1p and ORF2p, that associate with their parent transcript to form a ribonucleoprotein complex, an essential intermediate in L1 retrotransposition. Detailed mechanistic understanding of L1 retrotransposition is sparse, particularly with respect to the function of ORF1p, a coiled coil-mediated homotrimeric nucleic acid chaperone that can form tightly packed oligomers on nucleic acids. Here we studied three ORF1 proteins: a modern human wild type (111p), an inactive mutant (m15) wherein a single residue substitution within its coiled coil domain halts retrotransposition activity, and an active mutant (m14) with a largely modern coiled coil. We characterize ORF1p-nucleic acid interactions using an ∼ 8 kbp ssDNA molecule and measure its binding kinetics while the ssDNA is held at constant force. We show that ORF1p binds to ssDNA in a biphasic manner, in which a rapid decrease in ssDNA extension is followed by a relatively slow elongation of the ORF1p-ssDNA complex. The biphasic behavior observed here qualitatively mimics E. coli SSB binding dynamics to ssDNA. Therefore, for the first time, this study provides compelling evidence that ORF1p wraps ssDNA in multiple conformations. In addition to wrapping ssDNA, we show that the ORF1p-ssDNA complex undergoes extremely stable secondary compaction due to well-established protein-protein interactions. We present data which suggest that, although the ORF1p mutants can initially bind and wrap ssDNA in an identical manner as the wild type, the stability of the compact ORF1p-ssDNA complex is considerably weaker for the inactive m15 mutant when compared to 111p and the active m14 mutant.