DNA Structure Modulates the Oligomerization Properties of the AAV Initiator Protein Rep68

Rep68 is a multifunctional protein of the adeno-associated virus (AAV), a parvovirus that is mostly known for its promise as a gene therapy vector. In addition to its role as initiator in viral DNA replication, Rep68 is essential for site-specific integration of the AAV genome into human chromosome 19. Rep68 is a member of the superfamily 3 (SF3) helicases, along with the well-studied initiator proteins simian virus 40 large T antigen (SV40-LTag) and bovine papillomavirus (BPV) E1. Structurally, SF3 helicases share two domains, a DNA origin interaction domain (OID) and an AAA+ motor domain. The AAA+ motor domain is also a structural feature of cellular initiators and it functions as a platform for initiator oligomerization. Here, we studied Rep68 oligomerization in vitro in the presence of different DNA substrates using a variety of biophysical techniques and cryo-EM. We found that a dsDNA region of the AAV origin promotes the formation of a complex containing five Rep68 subunits. Interestingly, non-specific ssDNA promotes the formation of a double-ring Rep68, a known structure formed by the LTag and E1 initiator proteins. The Rep68 ring symmetry is 8-fold, thus differing from the hexameric rings formed by the other SF3 helicases. However, similiar to LTag and E1, Rep68 rings are oriented head-to-head, suggesting that DNA unwinding by the complex proceeds bidirectionally. This novel Rep68 quaternary structure requires both the DNA binding and AAA+ domains, indicating cooperativity between these regions during oligomerization in vitro. Our study clearly demonstrates that Rep68 can oligomerize through two distinct oligomerization pathways, which depend on both the DNA structure and cooperativity of Rep68 domains. These findings provide insight into the dynamics and oligomeric adaptability of Rep68 and serve as a step towards understanding the role of this multifunctional protein during AAV DNA replication and site-specific integration

Magnetic Properties of FePt Nanoparticles Prepared by a Micellar Method

FePt nanoparticles with average size of 9 nm were synthesized using a diblock polymer micellar method combined with plasma treatment. To prevent from oxidation under ambient conditions, immediately after plasma treatment, the FePt nanoparticle arrays were in situ transferred into the film-growth chamber where they were covered by an SiO2 overlayer. A nearly complete transformation of L10 FePt was achieved for samples annealed at temperatures above 700 °C. The well control on the FePt stoichiometry and avoidance from surface oxidation largely enhanced the coercivity, and a value as high as 10 kOe was obtained in this study. An evaluation of magnetic interactions was made using the so-called isothermal remanence (IRM) and dc-demagnetization (DCD) remanence curves and Kelly–Henkel plots (ΔM measurement). The ΔM measurement reveals that the resultant FePt nanoparticles exhibit a rather weak interparticle dipolar coupling, and the absence of interparticle exchange interaction suggests no significant particle agglomeration occurred during the post-annealing. Additionally, a slight parallel magnetic anisotropy was also observed. The results indicate the micellar method has a high potential in preparing FePt nanoparticle arrays used for ultrahigh density recording media