Critical contacts between HIV-1 integrase and viral DNA identified by structure-based analysis and photo-crosslinking

Analysis of the crystal structure of HIV‐1 integrase reveals a cluster of lysine residues near the active site. Using site‐directed mutagenesis and photo‐crosslinking we find that Lys156 and Lys159 are critical for the functional interaction of integrase with viral DNA. Mutation of Lys156 or Lys159...

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Bibliographic Details
Published in:The EMBO journal 1997-11, Vol.16 (22), p.6849-6859
Main Authors: Jenkins, Timothy M., Esposito, Dominic, Engelman, Alan, Craigie, Robert
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Binding Sites
Conserved Sequence
DNA Mutational Analysis
DNA, Viral - chemistry
DNA, Viral - metabolism
DNA-Binding Proteins - chemistry
DNA-Binding Proteins - metabolism
HIV Integrase - chemistry
HIV Integrase - metabolism
HIV-1
HIV-1 - enzymology
integrase
Integrases - genetics
Lysine - genetics
Models, Molecular
Molecular Sequence Data
photo-crosslinking
Protein Binding
Protein Conformation
protein-DNA interaction
Retroviridae - enzymology
Sequence Homology, Amino Acid
Species Specificity
Substrate Specificity
Virus Replication - genetics
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Summary:Analysis of the crystal structure of HIV‐1 integrase reveals a cluster of lysine residues near the active site. Using site‐directed mutagenesis and photo‐crosslinking we find that Lys156 and Lys159 are critical for the functional interaction of integrase with viral DNA. Mutation of Lys156 or Lys159 to glutamate led to a loss of both 3′ processing and strand transfer activities in vitro while maintaining the ability to interact with nonspecific DNA and support disintegration. However, mutation of both residues to glutamate produced a synergistic effect eliminating nearly all nonspecific DNA interaction and disintegration activity. In addition, virus containing either of these changes was replication‐defective at the step of integration. Photo‐crosslinking, using 5‐iododeoxyuracil‐substituted oligonucleotides, suggests that Lys159 interacts at the N7 position of the conserved deoxyadenosine adjacent to the scissile phosphodiester bond of viral DNA. Sequence conservation throughout retroviral integrases and certain bacterial transposases (e.g. Tn10/IS10) supports the premise that within those families of polynucleotidyl transferases, these residues are strategic for DNA interaction.
ISSN:0261-4189
1460-2075
1460-2075
DOI:10.1093/emboj/16.22.6849