Step-arrest mutants of phage Mu transposase. Implications in DNA-protein assembly, Mu end cleavage, and strand transfer.
We describe the isolation and characterization of Mu A variants arrested at specific steps of transposition. Mutations at 13 residues within the Mu A protein were analyzed for precise excision of Mu DNA in vivo. A subset of the defective variants (altered at Asp269, Asp294, Gly348, and Glu392) were tested in specific steps of transposition in vitro. It is possible that at least some residues of the Asp269-Asp294-Glu392 triad may have functional similarities to those of the conserved Asp-Asp-Glu motif found in several transposases and retroviral integrases. Mu A(D269V) is defective in high-order DNA-protein assembly, Mu end cleavage, and strand transfer. The assembly defect, but not the catalytic defect, can be overcome by precleavage of Mu ends. Mu A(E392A) can assemble the synaptic complex, but cannot cleave Mu ends. A mutation of Gly348 to aspartic acid within Mu A permits the uncoupling of cleavage and strand transfer activities. This mutant is completely defective in synaptic assembly and Mu end cleavage in presence of Mg2+. The assembly defect is alleviated by replacing Mg2+ with Ca2+. Some Mu end cleavage is observed with this mutant in the presence of Mn2+. When presented with precleaved Mu ends, Mu A(G348D) exhibits efficient strand transfer activity.
Kim
K
K
Department of Microbiology, University of Texas at Austin 78712.
Namgoong
S Y
SY
Jayaram
M
M
Harshey
R M
RM
D000595:Amino Acid Sequence; D010583:Bacteriophage mu / Q000201:enzymology* / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism*; D006868:Hydrolysis; D007477:Ions; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D009713:Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D017386:Sequence Homology, Amino Acid; D013569:Synapses / Q000378:metabolism; D019895:Transposases
D004279:DNA, Viral; D007477:Ions; D008345:Manganese; D009713:Nucleotidyltransferases; D019895:Transposases; D008274:Magnesium; D002118:Calcium