A covalent complex between retroviral integrase and nicked substrate DNA.
Purified retroviral integrase (IN) from avian sarcoma-leukosis viruses can appropriately process the termini of linear viral DNA, cleave host DNA in a sequence-independent manner, and catalyze integrative recombination; an exogenous source of energy is not required for these reactions. Using DNA substrates containing radioactive phosphate groups, we demonstrate that IN becomes covalently joined to the new 5' phosphate ends of DNA produced at sites of cleavage. Most of the phosphodiester linkages between IN and DNA involve serine, but some involve threonine. Computer-assisted alignment of 80 retroviral and retrotransposon IN sequences identified one serine that is conserved in all of these proteins and three less-conserved threonine residues. These results identify candidate active-site residues and provide support for the participation of a covalent IN-DNA intermediate in retroviral integration.
Katzman
M
M
Department of Medicine, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey 17033.
Mack
J P
JP
Skalka
A M
AM
Leis
J
J
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D009189:Avian Myeloblastosis Virus / Q000201:enzymology*; D001358:Avian Sarcoma Viruses / Q000201:enzymology*; D001483:Base Sequence; D001665:Binding Sites; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D004251:DNA Transposable Elements; D004279:DNA, Viral / Q000378:metabolism*; D019426:Integrases; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D010802:Phylogeny; D011485:Protein Binding; D012190:Retroviridae / Q000201:enzymology / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity
D000596:Amino Acids; D004251:DNA Transposable Elements; D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D004254:DNA Nucleotidyltransferases; D019426:Integrases