Characterization of the minimal DNA-binding domain of the HIV integrase protein.
The human immunodeficiency virus (HIV) integrase (IN) protein mediates an essential step in the retroviral lifecycle, the integration of viral DNA into human DNA. A DNA-binding domain of HIV IN has previously been identified in the C-terminal part of the protein. We tested truncated proteins of the C-terminal region of HIV-1 IN for DNA binding activity in two different assays: UV-crosslinking and southwestern blot analysis. We found that a polypeptide fragment of 50 amino acids (IN220-270) is sufficient for DNA binding. In contrast to full-length IN protein, this domain is soluble under low salt conditions. DNA binding of IN220-270 to both viral DNA and non-specific DNA occurs in an ion-independent fashion. Point mutations were introduced in 10 different amino acid residues of the DNA-binding domain of HIV-2 IN. Mutation of basic amino acid K264 results in strong reduction of DNA binding and of integrase activity.
Lutzke
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RA
Division of Molecular Biology, Netherlands Cancer Institute, Amsterdam.
Vink
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C
Plasterk
R H
RH
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D015139:Blotting, Southern; D015153:Blotting, Western; D003432:Cross-Linking Reagents; D004254:DNA Nucleotidyltransferases / Q000737:chemistry* / Q000378:metabolism*; D004279:DNA, Viral / Q000737:chemistry* / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology*; D019426:Integrases; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016415:Sequence Alignment; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D004279:DNA, Viral; D008345:Manganese; D004254:DNA Nucleotidyltransferases; D019426:Integrases; D008274:Magnesium