Directed integration of viral DNA mediated by fusion proteins consisting of human immunodeficiency virus type 1 integrase and Escherichia coli LexA protein.
We tested whether the selection of target sites can be manipulated by fusing retroviral integrase with a sequence-specific DNA-binding protein. A hybrid protein that has the Escherichia coli LexA protein fused to the C terminus of the human immunodeficiency virus type 1 integrase was constructed. The fusion protein, IN1-288/LA, retained the catalytic activities in vitro of the wild-type human immunodeficiency virus type 1 integrase (WT IN). Using an in vitro integration assay that included multiple DNA fragment as the target DNA, we found that IN1-288/LA preferentially integrated viral DNA into the fragment containing a DNA sequence specifically bound by LexA protein. No bias was observed when the LexA-binding sequence was absent, when the fusion protein was replaced by WT IN, or when LexA protein was added in the reaction containing IN1-288/LA. A majority of the integration events mediated by IN1-288/LA occurred within 30 bp of DNA flanking the LexA-binding sequence. The specificity toward the LexA-binding sequence and the distribution and frequency of target site usage were unchanged when the integrase component of the fusion protein was replaced with a variant containing a truncation at the N or C terminus or both, suggesting that the domain involved in target site selection resides in the central core region of integrase. The integration bias observed with the integrase-LexA hybrid shows that one effective means of altering the selection of DNA sites for integration is by fusing integrase to a sequence-specific DNA-binding protein.
Department of Molecular and Medical Pharmacology, UCLA School of Medicine 90095, USA.
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Goulaouic
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Chow
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D005240:Feasibility Studies; D015497:HIV-1 / Q000201:enzymology*; D006801:Humans; D019426:Integrases; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases; D013379:Substrate Specificity; D016662:Virus Integration / Q000235:genetics*; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D004254:DNA Nucleotidyltransferases; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D019426:Integrases; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D000099160:LexA Repressor Protein