Albany 2015:Book of Abstracts
June 9-13 2015
©Adenine Press (2012)
Oligomerization kinetics of ORF1p is correlated with LINE1 retrotransposition
ORF1 protein (p) is encoded by the long interspersed nuclear element-1 (LINE1) retrotransposon. LINE1 replicates by converting its transcript into genomic DNA, a mechanism that can also similarly process some host gene transcripts. LINE1 activity has thereby greatly expanded mammalian genomes during evolution and it causes variety of genetic changes in the modern human genome. The role of human ORF1p (hORF1p) in LINE1 retrotransposition is largely unknown, although it presumably involves its nucleic acid chaperone activity and its ability to oligomerize on nucleic acids. To better understand the molecular mechanism of hORF1p in LINE1-retrotransposition, we developed a novel method to characterize single-stranded DNA (ssDNA)-hORF1p interactions using single molecule stretching with optical tweezers. Here we examined the interactions of two hORF1p variants with ssDNA: hORF1-111p, the modern wild type (wt) protein and hORF1-151p, a hybrid of modern wt and a resuscitated ancestral hORF1p. Although the in vitro nucleic acid chaperone activities are indistinguishable in the two variants, 151p is inactive in an in vivo retrotransposition assay. We characterized three distinct binding kinetics for 111p and 151p with ssDNA. A fast kinetic fraction characterized by association and dissociation on a timescale of seconds, an intermediate fraction with a timescale of greater than one minute, which characterizes dissociation of the protein after the stretching force on DNA is released and a slow fraction with negligible dissociation on a timescale of tens of minutes. The fast fractions of both variants are converted to intermediate and slow fractions with time, consistent with protein oligomerization. However, oligomerization of 151p occurs two orders of magnitude slower than 111p. This result could explain the inactivity of 151p in retrotransposition and suggests that the oligomerization rate of ORF1p is important for retrotransposition.