Dissecting the roles of MuB in Mu transposition: ATP regulation of DNA binding is not essential for target delivery.
Collaboration between MuA transposase and its activator protein, MuB, is essential for properly regulated transposition. MuB activates MuA catalytic activity, selects target DNA, and stimulates transposition into the selected target site. Selection of appropriate target DNA requires ATP hydrolysis by the MuB ATPase. By fusing MuB to a site-specific DNA-binding protein, the Arc repressor, we generated a MuB variant that could select target DNA independently of ATP. This Arc-MuB fusion protein allowed us to test whether ATP binding and hydrolysis by MuB are necessary for stimulation of transposition into selected DNA, a process termed target delivery. We find that with the fusion proteins, MuB-dependent target delivery occurs efficiently under conditions where ATP hydrolysis is prevented by mutation or use of ADP. In contrast, no delivery was detected in the absence of nucleotide. These data indicate that the ATP- and MuA-regulated DNA-binding activity of MuB is not essential for target delivery but that activation of MuA by MuB strictly requires nucleotide-bound MuB. Furthermore, we find that the fusion protein directs transposition to regions of the DNA within 40-750 bp of its own binding site. Taken together, these results suggest that target delivery by MuB occurs as a consequence of the ability of MuB to stimulate MuA while simultaneously tethering MuA to a selected target DNA. This tethered-activator model provides an attractive explanation for other examples of protein-stimulated control of target site selection.
Schweidenback
Caterina T H
CT
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Baker
Tania A
TA
D000255:Adenosine Triphosphate / Q000502:physiology; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000502:physiology*; D018390:Gene Targeting; D007700:Kinetics; D011995:Recombination, Genetic; D019895:Transposases / Q000378:metabolism; D014764:Viral Proteins / Q000502:physiology*
D004268:DNA-Binding Proteins; C050938:Mu B protein, bacteriophage; D014764:Viral Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D019895:Transposases