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

Abstract

MuB, an ATP-dependent DNA-binding protein, is critical for the selection of target sites on the host chromosome during the phage Mu transposition. We developed a multichannel fluidic system to study the MuB-DNA interaction dynamics at the single DNA molecule level by total internal reflection fluorescence microscopy. We analyzed the distribution of MuB along DNA during the assembly and disassembly of MuB polymers on immobilized DNA molecules. The results reveal the absence of a significant correlation of MuB polymer distribution between the assembly and disassembly phases. These observations argue against a model in which MuB polymers on DNA represent a mixture of higher and lower affinity forms, with higher affinity forms being the first to appear and the last to disappear. Instead, assembly and disassembly of MuB polymers involve independent stochastic events. Additionally, we demonstrate that MuB disassembles from the polymer ends at a higher rate than from internal regions of the polymer and MuA stimulates MuB disassembly both at the polymer ends and internally.phage Mu ͉ recombination ͉ DNA-binding protein ͉ biomolecular patterning P hage Mu transposition is one of the most extensively exploited model systems for studying transpositional DNA recombination (1, 2). The reaction pathway is closely related to DNA integration of HIV-1 and other retroviruses (3) and VDJ recombination (4) in the development of the immune system. The phage Mu transposon integrates into the bacterial host chromosome at many sites with limited target sequence specificity. A puzzling aspect of Mu transposition is how the Mu DNA manages to distinguish itself from the target so as to avoid self-destructive autointegration into itself. Although Mu exhibits only limited target sequence specificity, DNA regions close to the Mu end sequence are poor targets for Mu transposition. This finding and related phenomena exhibited by many other transposons is called ''transposition target immunity.'' Mu target immunity involves the interplay between two phage-encoded proteins, MuA and MuB, and the formation of MuB distribution patterns along DNA molecules that define the regions preferred as target sites (5-7).The transposase MuA binds to the multiple copies of binding sequences located at the two Mu DNA ends, synapses the two ends, and forms a stable tetramer to which the two ends are bound. The resulting transposition-competent complex is called a ''transpososome.'' However, the complex does not promote efficient transposition to a new target site in the absence of MuB, an ATP-dependent DNA-binding protein. MuB activates Mu transpososomes for DNA strand transfer, the pair of transesterification reactions that insert the Mu DNA into target DNA. Thus, MuB-bound DNA acts as an efficient target for Mu transposon (8). Meanwhile, MuA stimulates the MuB ATPase and accelerates MuB dissociation from DNA. In the presence of a Mu end sequence on the DNA, MuA binds to this sequence and stimulates MuB dissociation from the neighboring DNA at a higher rate tha...

Analysis of phage Mu DNA transposition by whole-genome Escherichia coli tiling arrays reveals a complex relationship to distribution of target selection protein B, transcription and chromosome architectural elements

“…MuB binding is influenced by the A/T content of DNA in vitro (Greene and Mizuuchi 2004; Tan et al 2007; Ge and Harshey 2008). However, genes with a high frequency of CGG are preferred Mu targets in vivo (Manna et al 2005).…”

Transposable Phage Mu

“…3C) (34), which hydrolyzes ATP for target selection; both activities are stimulated by MuA (9). MuB forms ATP-dependent helical filaments, with or without DNA (34, 95), and has been reported to exhibit a tendency to form larger filaments on A/T-rich DNA (95, 96). Single-particle EM imaging of MuB assembled on DNA, found that MuB-ATP forms a solenoid-like filament, with DNA bound in the axial channel (34).…”

Congruence of in Vivo and in Vitro Insertion Patterns in Hot E. coli Gene Targets of Transposable Element Mu: Opposing Roles of MuB in Target Capture and Integration

“…Unlike a RecA filament which extends and unwinds DNA and promotes homology search and strand exchange within the filament, 31 the MuB filament does not appear to change DNA twist (our unpublished data; see also Ref. 22), and is apparently refractory to Mu integration inside the filament. Comparison of the data in Fig.…”