Controlling DNA degradation from a distance: a new role for the Mu transposition enhancer

Abstract

Summary Phage Mu is unique among transposable elements in employing a transposition enhancer. The enhancer DNA segment is the site where the transposase MuA binds and makes bridging interactions with the two Mu ends, interwrapping the ends with the enhancer in a complex topology essential for assembling a catalytically active transpososome. The enhancer is also the site at which regulatory proteins control divergent transcription of genes that determine the phage lysis-lysogeny decision. Here we report a third function for the enhancer - that of regulating degradation of extraneous DNA attached to both ends of infecting Mu. This DNA is protected from nucleases by a phage protein until Mu integrates into the host chromosome, after which it is rapidly degraded. We find that leftward transcription at the enhancer, expected to disrupt its topology within the transpososome, blocks degradation of this DNA. Disruption of the enhancer would lead to the loss or dislocation of two non-catalytic MuA subunits positioned in the transpososome by the enhancer. We provide several lines of support for this inference, and conclude that these subunits are important for activating degradation of the flanking DNA. This work also reveals a role for enhancer topology in phage development.

Repair of transposable phage Mu DNA insertions begins only when the E. coli replisome collides with the transpososome

“…1E; RecB − ), and degradation is delayed in a ClpX mutant, as reported earlier ( Fig. 1E; ClpX − ) (Au et al, 2006;Choi and Harshey, 2010;Choi et al, 2014b). A different integration assay showed additionally that the FD was processed to a short length in vivo ( Fig.…”

“…E; RecB − ), and degradation is delayed in a ClpX mutant, as reported earlier (Fig. E; ClpX − ) (Au et al ., ; Choi and Harshey, ; Choi et al ., ). A different integration assay showed additionally that the FD was processed to a short length in vivo (http://onlinelibrary.wiley.com/doi/10.1111/mmi.13061/suppinfo), similar to that seen in vitro with RecBCD (Fig.…”

“…Here, the ST intermediate is resolved without replication (Liebart et al, 1982;Akroyd and Symonds, 1983;Chaconas et al, 1983;Harshey, 1984), during which the FD is degraded concomitant with gap repair (Au et al, 2006). Using a convenient assay for monitoring FD degradation in vivo, we have learned that the first event in repair is removal of the FD by the RecBCD exonuclease (Choi et al, 2014a), whose entry past the N-protein block is controlled by the transpososome and facilitated by ClpX (Choi and Harshey, 2010;Choi et al, 2014b). In vitro experiments reveal that RecBCD action is required for stimulating endonucleolytic cleavage within the transpososome-protected DNA, leaving 4-nt flanks outside both Mu ends (Fig.…”

Transposition Behavior Revealed by High-Resolution Description of Pseudomonas Aeruginosa Saltovirus Integration Sites

“…A very small peak was visible within the first few hundred base-pairs in domain D1. The end of the domain D1 appeared to correspond to the location of the transposition enhancer region E in the phage Mu (reviewed in [ 9 ]). In the D9 domain, beyond position 36,500, the situation was unclear as most of the minor peak was probably an artifact due to the presence of the second Nsi I site, located 180 bp before the phage genome end.…”

“…Two phage-coded proteins, MuA and MuB, are essential for Mu transposition (reviewed in References [ 5 , 6 ]). MuA, a DDE recombinase, binds to the phage attachment sites (attL and attR) and to the phage transposition enhancer region whereas MuB, an AAA+ ATPase binds nonspecifically to the host chromosome and is therefore involved in the selection of insertion sites [ 6 , 7 , 8 , 9 ]. MuB assembles into a polymer that samples the chromosome, by assembly and disassembly involving MuA, until an appropriate site is located for the insertion of a bacteriophage genome [ 10 , 11 ].…”