RECENT FINDINGS: STRUCTURAL AND FUNCTIONAL STUDIES OF ORF1p - ORF1p is one of two L1 encoded proteins. Earlier studies by others of mouse ORF1p showed that it binds nucleic acids, acts as a nucleic acid chaperone, and forms trimers via a highly conserved coiled coil domain. However, the function of ORF1p in retrotransposition is largely unknown. We are using several approaches to examine this problem including analysis of the structural, biochemical, and biological effects of positive selection, which involved mainly the coiled coil domain (Boissinot, et al, Mol. Biol. Evol. 18: 2186). To do so we resuscitated an ORF1p from an extinct L1 family, L1Pa5, which is ancestral to the modern human(h) ORF1p of the currently active human L1Pa1 family. We also created mosaic versions of ORF1p that contain modern and ancestral regions, and other variants that were deleted of various domains. We extensively characterized these proteins with respect to retrotransposition and interaction with several mammalian host protein in vivo. In addition, we purified mg amounts of some the various of ORF1ps to homogeneity. We examined their various biophysical and biochemical properties in vitro including several assays that reflect their nucleic acid chaperone activity. We uncovered two novel properties of ORF1p in 2012: That hORF1p trimers can reversibly polymerize under the conditions required for high affinity nucleic acid binding, and that this property is involved in the second novel property of the protein, namely its biphasic effect of mismatched double-stranded nucleic acids, protecting it from dissociation (melting) at low concentrations, but melting it at high concentrations largely polymeric. A mismatched duplex is a proxy nucleic acid chaperone substrate. Thus, determining the biophysical basis the surprising and novel diphasic affect of the protein on this substrate is essential to our further progress in this area. Thus, in early 2013 we began a collaboration with Dr. Mark Williams to study these interactions by atomic force microscopy. This year, also implemented a new sensitive FRET assay for assessing nucleic acid chaperone activity under equilibrium conditions, carried out extensive mutational analysis of coiled coil domain in an attempt to clarify the biochemical effect of positive selection in this region of the protein, and determined that ORF1p is phosphorylated at a number of sites which we subjected to mutational analysis. The later two projects are nearing completion and we anticipate submitting our findings for publication later this year or early in 2014
| Furano, Anthony V; Cook, Pamela R (2016) The challenge of ORF1p phosphorylation: Effects on L1 activity and its host. Mob Genet Elements 6:e1119927 |
| Naufer, M Nabuan; Callahan, Kathryn E; Cook, Pamela R et al. (2016) L1 retrotransposition requires rapid ORF1p oligomerization, a novel coiled coil-dependent property conserved despite extensive remodeling. Nucleic Acids Res 44:281-93 |
| Naufer, M Nabuan; Furano, Anthony V; Williams, Mark C (2015) 12 Oligomerization kinetics of ORF1p is correlated with LINE1 retrotransposition. J Biomol Struct Dyn 33 Suppl 1:7-8 |
| Cook, Pamela R; Jones, Charles E; Furano, Anthony V (2015) Phosphorylation of ORF1p is required for L1 retrotransposition. Proc Natl Acad Sci U S A 112:4298-303 |
| Callahan, Kathryn E; Hickman, Alison B; Jones, Charles E et al. (2012) Polymerization and nucleic acid-binding properties of human L1 ORF1 protein. Nucleic Acids Res 40:813-27 |
