Abstract
MuB, a protein essential for replicative DNA transposition by the bacteriophage Mu, is an ATPase that assembles into a polymeric complex on DNA. We used total internal reflection fluorescence microscopy to observe the behavior of MuB polymers on single molecules of DNA. We demonstrate that polymer assembly is initiated by a stochastic nucleation event. After nucleation, polymer assembly occurs by a mechanism involving the sequential binding of small units of MuB. MuB that bound to A/T-rich regions of the DNA assembled into large polymeric complexes. In contrast, MuB that bound outside of the A/T-rich regions failed to assemble into large oligomeric complexes. Our data also show that MuB does not catalyze multiple rounds of ATP hydrolysis while remaining bound to DNA. Rather, a single ATP is hydrolyzed, then MuB dissociates from the DNA. Finally, we show that "capping" of the enhanced green fluorescent protein-MuB polymer ends with unlabeled MuB dramatically slows, but does not halt, dissociation. This suggests that MuB dissociation occurs through both an end-dependent mechanism and a slower mechanism wherein subunits dissociate from the polymer interior.
Highlights
MuB performs multiple functions during transposition, including stimulating assembly of the transpososomes, stimulating the MuA catalyzed DNA processing reactions, influencing the selection of the DNA target site, and regulating the disassembly of the transpososome (10 –13)
We demonstrate that polymer assembly is initiated by a nucleation event in which a small unit of MuB binds to A/T-rich DNA in a relatively stable state
Of Single MuB Target Complexes—We have reported the development of a total internal reflection fluorescence microscope system for monitoring the behavior of single enhanced green fluorescent protein (EGFP)-labeled MuB polymers bound to DNA [17]
Summary
Vol 279, No 16, Issue of April 16, pp. 16736 –16743, 2004 Printed in U.S.A. Visualizing the Assembly and Disassembly Mechanisms of the MuB Transposition Targeting Complex*. We have demonstrated that MuB forms a large oligomer on DNA and that these oligomers were tightly bound to A/T-rich sequences [17] These same A/T-rich regions were preferentially used as transposition targets by the MuA transposase. We demonstrate that polymer assembly is initiated by a nucleation event in which a small unit of MuB binds to A/T-rich DNA in a relatively stable state. This is followed by the sequential net addition of small subunits to the growing complex. We propose that the MuB polymers can disassemble by at least two pathways: either by the dissociation of MuB from the ends of the individual polymers or by the relatively slow dissociation of MuB subunits from the interior of the polymeric complex
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