Role of the His-Cys finger of Moloney murine leukemia virus integrase protein in integration and disintegration.
Retroviral integrases mediate site-specific endonuclease and transesterification reactions in the absence of exogenous energy. The basis for the sequence specificity in these integrase-viral DNA recognition processes is unknown. Structural analogs of the disintegration substrate were made to analyze the disintegration reaction mechanism for the Moloney murine leukemia virus (M-MuLV) integrase (IN). Modifications in the target DNA portion of the disintegration substrate decreased enzymatic activity, while substitution of the highly conserved CA in the viral long terminal repeat portion had no effect on activity. The role of the His-Cys finger region in catalysis was addressed by N-ethylmaleimide (NEM) modification of the cysteine residues of M-MuLV IN as well as by mutations. Both integration activities, 3' processing, and strand transfer, were completely inhibited by NEM modification of M-MuLV IN, while disintegration activity was only partially sensitive. However, structural analogs of the disintegration substrates that were modified in the target DNA and had the conserved CA removed were not active with NEM-treated M-MuLV IN. In addition, mutants made in the His-Cys region of M-MuLV IN were examined and found to also be completely blocked in integration but not disintegration activity. These data suggest that the domains of M-MuLV IN that are required for the forward integration reaction substrate differ from those required for the reverse disintegration reaction substrate.
Jonsson
C B
CB
Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry, Piscataway, New Jersey 08854.
Roth
M J
MJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D003545:Cysteine; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D006639:Histidine; D019426:Integrases; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000201:enzymology* / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D013379:Substrate Specificity; D016662:Virus Integration
D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D006639:Histidine; D004254:DNA Nucleotidyltransferases; D019426:Integrases; D003850:Deoxyribonuclease I; D003545:Cysteine; D005033:Ethylmaleimide