Localization of ASV Integrase-DNA Contacts by Site-Directed Crosslinking and their Structural Analysis

We applied crosslinking techniques as a first step in preparation of stable avian sarcoma virus (ASV) integrase (IN)-DNA complexes for crystallographic investigations. These results were then compared with the crystal structures of the prototype foamy virus (PFV) intasome and with published data for other retroviral IN proteins.Photoaffinity crosslinking and site-directed chemical crosslinking were used to localize the sites of contacts with DNA substrates on the surface of ASV IN. Sulfhydryl groups of cysteines engineered into ASV IN and amino-modified nucleotides in DNA substrates were used for attachment of photocrosslinkers. Analysis of photocrosslinking data revealed several specific DNA-protein contacts. To confirm contact sites, thiol-modified nucleotides were introduced into oligo-DNA substrates at suggested points of contact and chemically crosslinked to the cysteines via formation of disulfide bridges. Cysteines incorporated in positions 124 and 146 in the ASV IN core domain were shown to interact directly with host and viral portions of the Y-mer DNA substrate, respectively. Crosslinking of an R244C ASV IN derivative identified contacts at positions 11 and 12 on both strands of viral DNA. The most efficient disulfide crosslinking was observed for complexes of the ASV IN E157C and D64C derivatives with linear viral DNA substrate carrying a thiol-modified scissile phosphate.Analysis of our crosslinking results as well as published results of retroviral IN protein from other laboratories shows good agreement with the structure of PFV IN and derived ASV, HIV, and MuLV models for the core domain, but only partial agreement for the N- and C-terminal domains. These differences might be explained by structural variations and evolutionary selection for residues at alternate positions to perform analogous functions, and by methodological differences: i.e., a static picture of a particular assembly from crystallography vs. a variety of interactions that might occur during formation of functional IN complexes in solution

A framework for reviewing the trade-offs between, renewable energy, food, feed and wood production at a local level

High fuel prices and concerns about energy security and anthropogenic climate change are encouraging a transition towards a low carbon economy. Although energy policy is typically set at a national level, tools are needed for people to engage with energy policy at regional and local levels, and to guide decisions regarding land use, distributed generation and energy supply and demand. The aim of this paper is to develop a per-capita approach to renewable energy demand and supply within a landscape and to illustrate the key trade-offs between renewable energy, food, (animal) feed and wood production. The chosen case study area (16,000 ha) of Marston Vale, England is anticipated to have a population density midway between that for England and the UK. The daily per capita demand for energy for heat (31 kWh), transport (34 kWh) and electricity (15 kWh) when combined (80 kWh) was seven-fold higher than the combined demand for food (2 kWh), animal feed (6 kWh), and wood (4 kWh). Using described algorithms, the combined potential energy supply from domestic wind and photovoltaic panels, solar heating, ground-source heat, and municipal waste was limited (<10 kWh p−1d−1). Additional electricity could be generated from landfill gas and commercial wind turbines, but these have temporal implications. Using a geographical information system and the Yield-SAFE tree and crop yield model, the capacity to supply bioethanol, biodiesel, and biomass, food, feed and wood was calculated and illustrated for three land-use scenarios. These scenarios highlight the limits on meeting energy demands for transport (33%) and heat (53%), even if all of the arable and grassland area was planted to a high yielding crop like wheat. The described framework therefore highlights the major constraints faced in meeting current UK energy demands from land-based renewable energy and the stark choices faced by decision ma