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Functional and structural characterization of the integrase from the prototype foamy virus. Nucleic Acids Res
- 2009
"... from the prototype foamy virus ..."
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Localization of ASV Integrase-DNA Contacts by Site- Directed Crosslinking and their Structural Analysis
"... Background: 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 publis ..."
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Background: 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. Methodology/Results: 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 aminomodified 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. Conclusion: Analysis of our crosslinking results as well as published results of retroviral IN protein from other laboratories
Entering a New Phase: Ways & Means Using Solvent Halide Ions in Protein Structure Determination
"... fraction data. Bromine has one electron more than sele-nium and its absorption edge at 0.92 AÊ is similar to that of selenium at 0.98 AÊ. In the same way that selenomethi-onine (SeMet) is routinely used to determine protein structures using multiwavelength anomalous diffraction ..."
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fraction data. Bromine has one electron more than sele-nium and its absorption edge at 0.92 AÊ is similar to that of selenium at 0.98 AÊ. In the same way that selenomethi-onine (SeMet) is routinely used to determine protein structures using multiwavelength anomalous diffraction
A Possible Role for the Asymmetric C-Terminal Domain Dimer of Rous Sarcoma Virus Integrase in Viral DNA Binding
, 2012
"... Integration of the retrovirus linear DNA genome into the host chromosome is an essential step in the viral replication cycle, and is catalyzed by the viral integrase (IN). Evidence suggests that IN functions as a dimer that cleaves a dinucleotide from the 39 DNA blunt ends while a dimer of dimers (t ..."
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Integration of the retrovirus linear DNA genome into the host chromosome is an essential step in the viral replication cycle, and is catalyzed by the viral integrase (IN). Evidence suggests that IN functions as a dimer that cleaves a dinucleotide from the 39 DNA blunt ends while a dimer of dimers (tetramer) promotes concerted integration of the two processed ends into opposite strands of a target DNA. However, it remains unclear why a dimer rather than a monomer of IN is required for the insertion of each recessed DNA end. To help address this question, we have analyzed crystal structures of the Rous sarcoma virus (RSV) IN mutants complete with all three structural domains as well as its two-domain fragment in a new crystal form at an improved resolution. Combined with earlier structural studies, our results suggest that the RSV IN dimer consists of highly flexible N-terminal domains and a rigid entity formed by the catalytic and C-terminal domains stabilized by the well-conserved catalytic domain dimerization interaction. Biochemical and mutational analyses confirm earlier observations that the catalytic and the C-terminal domains of an RSV IN dimer efficiently integrates one viral DNA end into target DNA. We also show that the asymmetric dimeric interaction between the two C-terminal domains is important for viral DNA binding and subsequent catalysis, including concerted integration. We propose that the asymmetric C-terminal domain dimer serves
CONTENT ALERTS
, 2002
"... This article cites 42 articles, 21 of which can be accessed free ..."
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