A subsequence-specific DNA-binding domain resides in the 13 kDa amino terminus of the bacteriophage Mu transposase protein.
We have previously reported that the 13 kDa amino terminus of the 70 kDa bacteriophage D108 transposase protein (A gene product) contains a two-component, sequence-specific DNA-binding domain which specifically binds to the related bacteriophage Mu's right end (attR) in vitro. To extend these studies, we examined the ability of the 13 kDa amino terminus of the Mu transposase protein to bind specifically to Mu attR in crude extracts. Here we report that the Mu transposase protein also contains a Mu attR specific DNA-binding domain, located in a putative alpha-helix-turn-alpha-helix region, in the amino terminal 13 kDa portion of the 70 kDa transposase protein as part of a 23 kDa fusion protein with beta-lactamase. We purified for this attR-specific DNA-binding activity and ultimately obtained a single polypeptide of the predicted molecular weight for the A'--'bla fusion protein. We found that the pure protein bound to the Mu attR site in a different manner compared with the entire Mu transposase protein as determined by DNase I-footprinting. Our results may suggest the presence of a potential primordial DNA-binding site (5'-PuCGAAA-3') located several times within attR, at the ends of Mu and D108 DNA, and at the extremities of other prokaryotic class II elements that catalyze 5 base pair duplications at the site of element insertion. The dissection of the functional domains of the related phage Mu and D108 transposase proteins will provide clues to the mechanisms and evolution of DNA transposition as a mode of mobile genetic element propagation.
Tolias
P P
PP
Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada.
DuBow
M S
MS
D010583:Bacteriophage mu / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D004279:DNA, Viral; D003850:Deoxyribonuclease I; D008970:Molecular Weight; D009713:Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism; D019895:Transposases; D014760:Viral Fusion Proteins
D004279:DNA, Viral; D014760:Viral Fusion Proteins; D009713:Nucleotidyltransferases; D019895:Transposases; D003850:Deoxyribonuclease I