RECENT FINDINGS: STRUCTURAL AND FUNCTIONAL STUDIES OF ORF1p - ORF1p is one of two L1 encoded proteins that are essential for retrotransposition. We earlier showed that primate ORF1p (like that in mouse) is a coiled coil mediated trimer that has nucleic acid binding and chaperone activity. However, the function of ORF1p in retrotransposition is largely unknown. This past year we made two important advances in our studies of ORF1p: (1) We showed that L1 activity requires phosphorylation of highly conserved proline dependent phosphokinase (PDPK) sites. We also showed that ORF1p expressed in HeLa cells can bind Pin1, a protein that serves a critical function in phosphorylation mediated events by catalyzing cis / trans isomerization of proline at phosphorylated PDPK sites. Reversible protein phosphorylation is effected by interacting cascades of protein kinases, phosphatases, and ancillary proteins, and is a mainstay in the regulation and coordination of many basic biological processes. Therefore, demonstrating phosphorylation-dependence of L1 activity substantially enlarged our knowledge of the scope of L1 / host interaction. (2) In collaboration with the Williams laboratory at Northeastern University, we used single molecule studies to show that an ORF1p with a mutated coiled coil that is inactive in retrotransposition, but exhibits no significant biochemical differences from an active ORF1p with a non-mutated coiled coil, forms stably bound oligomers on ssDNA at <1/10th the rate of active proteins. Thus, oligomerization rate is a novel and critical coiled coil-dependent parameter of ORF1p activity. We also showed that this property has been conserved, despite extensive remodeling of the coiled coil during the last 25 Myr of primate evolution, thus rationalizing the repeated episodes of positive selection that typifies coiled coil evolution.
| 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 |
