Mutations in domain III alpha of the Mu transposase: evidence suggesting an active site component which interacts with the Mu-host junction.
A series of point mutations was constructed in domain IIIalpha of the Mu A protein. The mutant transposases were purified and assayed for their ability to promote various aspects of the in vitro Mu DNA strand transfer reaction. All mutants with discernable phenotypes were inhibited in stable synapsis (Type 0 or Type 1 complex formation). In contrast, these mutant proteins were capable of LER formation (a transient early reaction intermediate in which the Mu left and right ends have been synapsed with the enhancer), at levels comparable to wild-type transposase. These proteins therefore comprise a novel class of transposase mutants, which are specifically inhibited in stable transpososome assembly. The defect in these proteins was also uniformly suppressed by either Mn2+, or the Mu B protein in the presence of ATP and target DNA. Striking phenotypic similarities were recognized between the domain IIIalpha transposase mutant characteristics noted above, and those for substrate mutants carrying a terminal base-pair substitution at the point of cleavage on the donor molecule. This phenotypic congruence suggests that the alterations in either protein or DNA are exerting an effect on the same step of the reaction i.e., engagement of the terminal nucleotide by the active site. We suggest that domain IIIalpha of the transposase comprises the substrate binding pocket of the active site which interacts with the Mu-host junction.
Naigamwalla
D Z
DZ
Department of Biochemistry, University of Western Ontario, London, Ontario, N6A 5C1, Canada.
Coros
C J
CJ
Wu
Z
Z
Chaconas
G
G
D000595:Amino Acid Sequence; D010583:Bacteriophage mu / Q000201:enzymology / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D004269:DNA, Bacterial / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010641:Phenotype; D017354:Point Mutation / Q000235:genetics*; D011499:Protein Processing, Post-Translational / Q000235:genetics; D017434:Protein Structure, Tertiary; D019895:Transposases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D004269:DNA, Bacterial; D004279:DNA, Viral; D014764:Viral Proteins; C114697:mu transposase; D019895:Transposases