Mapping features of HIV-1 integrase near selected sites on viral and target DNA molecules in an active enzyme-DNA complex by photo-cross-linking.
The virally encoded integrase protein carries out retroviral integration, and to do so, it must make specific interactions with both viral and target DNA sequences. The retroviral integrase has three domains: an amino-terminal region of about 50 amino acids that contains a zinc finger-like motif, a tightly folded, phylogenetically conserved core domain that contains the active site, and a carboxy-terminal domain that can bind DNA in a nonspecific manner. The complete roles of the amino- and carboxyl-terminal domains have not yet been determined, but they appear to participate in multimerization and nonspecific or target DNA binding, respectively. The number and identity of integrase's DNA binding sites have been difficult to determine by conventional mutagenesis studies. In this report, we describe a photo-cross-linking approach to address these issues. Our findings suggest that HIV-1 integrase contacts with conserved features of the viral DNA end are likely to be mediated by residues in two peptides within the conserved core domain. Additional cross-links were seen between viral DNA and the carboxy-terminal DNA binding domain. Numerous sites in integrase, including peptides in each of the three domains, could be cross-linked to target DNA features. Integrase is known to function as a multimer, and it remains to be determined which specific contacts are in cis or trans with respect to the active site.
Heuer
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Department of Biochemistry, Program in Cancer Biology, Stanford University Medical Center, California 94305-5428, USA.
Brown
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PO
D001386:Azides; D002384:Catalysis; D017124:Conserved Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism*; D003596:Cytosine / Q000031:analogs & derivatives; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D019427:HIV Integrase / Q000737:chemistry / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D007202:Indicators and Reagents; D010449:Peptide Mapping / Q000379:methods*; D010777:Photochemistry; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D014466:Ultraviolet Rays
D001386:Azides; D003432:Cross-Linking Reagents; D004279:DNA, Viral; D007202:Indicators and Reagents; C008143:5-iodocytosine; D003596:Cytosine; D019427:HIV Integrase