A substitution in rous sarcoma virus integrase that separates its two biologically relevant enzymatic activities.
Retroviral integrase prepares viral DNA for integration by removing 2 nucleotides from each end of unintegrated DNA in a reaction referred to as processing. However, it has been known since the processing assay was first described that avian integrases frequently nick 3 nucleotides, as well as 2 nucleotides, from viral DNA ends when reaction mixtures contain Mn2+. We now report that specificity for the biologically relevant "-2" site is enhanced when the serine at amino acid 124 of Rous sarcoma virus (RSV) integrase is replaced by alanine, valine, glycine, lysine, or aspartate. The protein with a serine-to-aspartate substitution exhibited especially high fidelity for the correct site, as evidenced by a ratio of -2 nicks to -3 nicks that was more than 40-fold greater than that for the wild-type enzyme in reactions with Mn2+. Even with Mg2+, the substituted proteins exhibited greater specificity than the wild type, especially the S124D protein. Moreover, this protein was more efficient than the wild type at processing viral DNA ends. Unexpectedly, however, the S124D protein was significantly impaired at catalyzing the insertion of viral DNA ends in reactions with Mn2+ and joining was undetectable in reactions with Mg2+. Thus, the S124D protein has separated the processing and joining activities of integrase. Similar results were found for human immunodeficiency virus integrase with the analogous substitution. No proteins with comparable properties have been described. Moreover, RSV virions containing integrase with the S124D mutation were unable to replicate in cell cultures. Together, these data suggest that integrase has evolved to have submaximal processing activity so that it can also catalyze DNA joining.
Konsavage
Wesley M
WM
Department of Microbiology and Immunology, The Milton S. Hershey Medical Center, P.O. Box 850, Mail Code H036, Hershey, PA 17033-0850, USA.
Burkholder
Stephen
S
Sudol
Malgorzata
M
Harper
Amy L
AL
Katzman
Michael
M
D019943:Amino Acid Substitution; D001358:Avian Sarcoma Viruses / Q000201:enzymology*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology; D019426:Integrases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012190:Retroviridae / Q000201:enzymology; D016662:Virus Integration; D014779:Virus Replication / Q000235:genetics
D011994:Recombinant Proteins; D008345:Manganese; D019426:Integrases; D008274:Magnesium