Two mutations of phage mu transposase that affect strand transfer or interactions with B protein lie in distinct polypeptide domains.
Two mutations within the transposase (the A protein) gene of phage Mu with distinct effects on DNA transposition have been studied. The first mutation maps to the central domain (domain II) of A, a protein consisting of three major structural domains. The variant protein is normal in synapsis and cleavage of Mu ends but is temperature-sensitive in the strand transfer reaction, joining the Mu ends to target DNA. The second mutation is a deletion at the C terminus (within domain III); on the basis of genetic studies, the mutant protein is predicted to have lost the ability to interact with the Mu B protein. The B protein, in conjunction with A, promotes efficient intermolecular transposition, while inhibiting intramolecular transposition. We show that the purified mutant protein is proficient in intramolecular, but not intermolecular transposition in vitro. The interactions between A and B proteins have been followed by a proteolysis assay. The chymotrypsin sensitivity of the interdomainal Phe221-Ser222 peptide bond within the bidomainally organized B protein is exquisitely modulated by ATP, DNA and A protein. The sensitive or "open" state of this bond in native B protein becomes partially "open" upon binding of ATP by B, attains a "closed" or resistant configuration upon binding of DNA in presence of ATP, and is rendered "open" again upon addition of the A protein. In this test for the interaction of A protein with B protein-DNA complex, the domain II mutant behaves like wild-type A protein. However, the domain III mutant fails to restore chymotrypsin susceptibility of the Phe221-Ser222 bond.
Leung
P C
PC
Department of Microbiology, University of Texas, Austin 78712.
Harshey
R M
RM
D010583:Bacteriophage mu / Q000201:enzymology / Q000235:genetics*; D002872:Chromosome Deletion; D002918:Chymotrypsin; D002942:Circular Dichroism; D004279:DNA, Viral / Q000235:genetics / Q000648:ultrastructure; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005814:Genes, Viral; D014644:Genetic Variation; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D009154:Mutation; D009713:Nucleotidyltransferases / Q000235:genetics*; D010449:Peptide Mapping; D010957:Plasmids; D011487:Protein Conformation; D015183:Restriction Mapping; D019895:Transposases; D015678:Viral Structural Proteins / Q000235:genetics*
D004279:DNA, Viral; D046911:Macromolecular Substances; D015678:Viral Structural Proteins; D009713:Nucleotidyltransferases; D019895:Transposases; D002918:Chymotrypsin