ROLE OF THE HIS-CYS FINGER OF MOLONEY MURINE LEUKEMIA-VIRUS INTEGRASEPROTEIN IN INTEGRATION AND DISINTEGRATION

Retroviral integrases mediate site-specific endonuclease and transeste rification reactions in the absence of exogenous energy. The basis for the sequence specificity in these integrase-viral DNA recognition pro cesses is unknown. Structural analogs of the disintegration substrate were made to analyze the disintegration reaction mechanism for the Mol oney murine leukemia virus (M-MuLV) integrase (IN). Modifications in t he target DNA portion of the disintegration substrate decreased enzyma tic activity, while substitution of the highly conserved CA in the vir al long terminal repeat portion had no effect on activity. The role of the His-Cys finger region in catalysis was addressed by N-ethylmaleim ide (NEM) modification of the cysteine residues of M-MuLV IN as well a s 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 wi th NEM-treated M-MuLV IN. In addition, mutants made in the His-Cys reg ion of M-MuLV IN were examined and found to also be completely blocked in integration but not disintegration activity. These data suggest th at the domains of M-MuLV IN that are required for the forward integrat ion reaction substrate differ from those required for the reverse disi ntegration reaction substrate.