Visualizing the Assembly and Disassembly Mechanisms of the MuB Transposition Targeting Complex

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.DNA transposition is used by many organisms to regulate the dispersion of their genetic material throughout a host cell population (1, 2). The bacteriophage Mu transposition reaction is initiated by the binding of the transposase protein MuA to specific sites at the ends of the phage genome (3). MuA then assembles into a series of nucleoprotein complexes, collectively referred to as transpososomes, that are responsible for catalyzing the DNA processing reactions required to complete transposition (4 -7). A second protein, the ATPase MuB, is also required to complete the transposition reaction (8, 9).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). MuB does not share extensive sequence similarity with any other known protein.However, like many other ATPases, MuB does contain two conserved amino acid motifs, Walker A and B boxes, that are essential for the binding and hydrolysis of nucleotide triphosphates (14, 15). In the presence of ATP, MuB assembles into large oligomeric complexes on DNA, making the bound DNA an efficient target for strand transfer (16,17). MuB accumulates on DNA that is not bound by MuA, resulting in a strong preference for transposition to occur at DNA sites at least 10 -20 kb from MuA-bound regions (12,18). This phenomenon is called target immunity and provides a critical mechanism for Mu to prevent disruption of its genome through an aberrant transposition event.We have established a system that allows us to observe single complexes of MuB bound to an immobilized molecule of DNA (17). We have also demonstrated that MuB forms a large oligomer on DNA and that these oligomers were tightly bound to A/T-rich sequences (17)...

DNA transposition target immunity and the determinants of the MuB distribution patterns on DNA

“…First, we observe a 5-fold decrease in the apparent dissociation rate constant when we compare the rate constant distribution in buffer containing saturating unlabeled MuB (red histogram) with the rate constant distribution in plain buffer without MuB (black histogram). The unlabeled MuB acts as a polymer end-capper and significantly slows down the disassembly of the fluorescent MuB target complexes, confirming previously reported observations (9). However, even higher concentrations of unlabeled MuB do not completely block the exchange of labeled MuB by unlabeled molecules.…”

“…The mean value of the dissociation rate constants among target DNA molecules was 0.50 min Ϫ1 with a half distribution width of 0.074 min Ϫ1 . The apparent dissociation rate constant observed is consistent with previous reports (9,11). Because the DNA-bound MuB exists in polymers of heterogeneous sizes, the dissociation curve should not necessarily fit to a singleexponential decay.…”

“…The distribution pattern of DNA-bound MuB evolved relatively slowly, taking many minutes. With time, some of the smaller polymers grew even smaller and disappeared, whereas others grew larger; after a longer incubation, such as 30 min, the MuB distribution pattern of individual DNA molecules became alike (9).…”

“…Previous experiments indicated that the MuB clusters observed represent a polymeric form of MuB assembled on DNA, and MuB dissociation predominantly takes place at the polymer ends (9). The preferential dissociation of MuB from ends is easily understandable because MuB polymerization must involve cooperative interactions among neighboring monomers, and such stabilizing interactions are partially missing at the polymer ends.…”

“…We used total internal reflection fluorescence (TIRF) microscopy with surface-immobilized phage -DNA to visually monitor the association/dissociation dynamics of fluorescently labeled MuB under various conditions (7,(9)(10)(11). At saturating concentrations of MuB in the presence of ATP, -DNA was evenly coated with MuB.…”