Characterization of functionally important sites in the bacteriophage Mu transposase protein.
The 663 amino acid Mu transposase protein is absolutely required for Mu DNA transposition. Mutant proteins were constructed in vitro in order to locate regions of transposase that may be important for the catalysis of DNA transposition. Deletions in the A gene, which encodes the transposase, yielded two stable mutant proteins that aid in defining the end-specific DNA-binding domain. Linker insertion mutagenesis at eight sites in the Mu A gene generated two proteins, FF6 and FF14 (resulting from two and four amino acid insertions, respectively, at position 408), which were thermolabile for DNA binding in vitro at 43 degrees C. However, transposition activity in vivo was severely reduced for all mutant proteins at 37 degrees C, except those with insertions at positions 328 and 624. In addition, site-specific mutagenesis was performed to alter tyrosine 414, which is situated in a region that displays amino acid homology to the active sites of a number of nicking/closing enzymes. Tyrosine 414 may reside within an important, yet non-essential, site of transposase, as an aspartate-substituted protein had a drastically reduced frequency of transposition, while the remaining mutants yielded reduced, but substantial, frequencies of microMu transposition in vivo.
Ulycznyj
P I
PI
Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada.
Forghani
F
F
DuBow
M S
MS
D000595:Amino Acid Sequence; D010583:Bacteriophage mu / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D004279:DNA, Viral / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009713:Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D019895:Transposases; D014443:Tyrosine / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D004279:DNA, Viral; D014764:Viral Proteins; D014443:Tyrosine; D009713:Nucleotidyltransferases; D019895:Transposases