Functional domains of Moloney murine leukemia virus integrase defined by mutation and complementation analysis.
Retroviral integrases perform two catalytic steps, 3' processing and strand transfer, that result in the stable insertion of the retroviral DNA into the host genome. Mutant M-MuLV integrases were constructed to define the functional domains important for 3' processing, strand transfer, and disintegration by in vitro assays. N-terminal mutants had no detectable 3' processing activity, and only one mutant which lacks the HHCC domain, Ndelta105, had strand transfer activity. Strand transfer mediated by Ndelta105 showed preference for one site in the target DNA. Disintegration activity of N-terminal mutants decreased only minimally. In contrast, all C-terminal mutants truncated by more than 28 amino acids had no integration or disintegration activity. Activity on a single-strand disintegration substrate did not require a functional HHCC domain but did require most of the C-terminal region. Complementation analysis found that the HHCC region alone was able to function in trans to a promoter containing only the DD(35)E and C-terminal regions and to enhance integration site selection. Increasing the reducing conditions or adding the HHCC domain to Ndelta105 reaction mixtures restored the wild-type strand transfer activity and range of target sites. The reducing agent affected Cys-209 in the DD(35)E region. The presence of C-209 was required for complementation of Ndelta105 by the HHCC region.
Jonsson
C B
CB
Department of Biochemistry, Robert W. Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854, USA.
Donzella
G A
GA
Gaucan
E
E
Smith
C M
CM
Roth
M J
MJ
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D005816:Genetic Complementation Test; D019426:Integrases; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D016296:Mutagenesis; D017384:Sequence Deletion; D016662:Virus Integration
D017931:DNA Primers; D004254:DNA Nucleotidyltransferases; D019426:Integrases