Chapter 31 : Transposable Phage Mu

One transposition mechanism, two pathways for resolution of the ST intermediate. The chemical steps of cleavage and ST are the same in both the infection and lytic phases of transposition. (A) In the infection phase, the linear donor Mu genome is converted into a noncovalently closed circle, joined by the MuN protein (purple ovals, ends shown unjoined for clarity); E. coli genome is the target. The ST intermediate formed during intermolecular transposition is resolved by removal of the FD, and repair of the 5-bp gaps in the target by limited replication at the host–Mu junction. (B) In the lytic phase, Mu is part of the covalently closed circular E. coli genome. The ST intermediate formed during intramolecular transposition is resolved by replication across Mu.

Disintegration: true and pseudo reversal. Ovals represent the transposase active sites. (A) True reversal refers to restoration of the original target configuration by a cis nucleophilic attack of the target 3′-OHs on the target–host junction generated during ST. L and R refer to the left and right ends of Mu. (B) In pseudo reversal, the target is rearranged (imagine unpairing the 5 bp in A, and flipping the DNA through 180°), so that the target nucleophiles attack the host–target junction in trans, resulting in hairpin products. (C) True reversal is more facile if the target carries a mismatch (left) indicated by an unpaired base pair, or if only a single end undergoes ST within the transpososome (right). See text for details.

DNA and protein requirements for transposition. (A) Arrangement of MuA binding sites at the L (L1–L3) and R (R1–R3) ends of the Mu genome, and within the Mu enhancer E (O1–O3). E is positioned closer to the L end on the Mu genome, and is also labeled O because of its dual function as an operator that regulates lysis/lysogeny decision ( 8 ). HU and IHF bind within L and E as shown. FD on either side of the Mu genome is packaged into virions. (B) Domain and subdomain organization of MuA as assigned by partial proteolysis ( 140 ). NMR and crystal structures for the individual subdomains (except IIIβ) are available ( 9 , 62 , 141 ). The subdomain IIβ was observed in crystal structures ( 142 ), while IIIα and IIIβ were delineated by mutagenesis and functional studies ( 9 ). BAN stands for DNA-binding and nuclease function ( 122 ). See reference 143 for an insertion mutagenesis study across the domains. (C) Domain organization of MuB, as assigned by partial proteolysis ( 144 ). An NMR structure for the C-terminal domain is available ( 145 ). An AAA+ ATPase function spanning residues in both domains was deduced by bioinformatics, and supported by mutagenesis ( 34 ). Both domains also contribute to nonspecific DNA binding ( 9 , 34 , 35 ). A patch of positively charged residues (KKK) on the C-terminal domain likely interacts with MuA to trigger ATP hydrolysis ( 34 , 35 ). The conformation of the hinge region between the domains is exquisitely modulated by ATP, DNA, and A protein, as judged by its sensitivity to proteolysis ( 146 ).

DNA–protein transitions within the MuA active sites. (A) Terminal base pairs at the Mu ends (L1 and R1 sites) and its adjoining FD are engaged within the MuA active sites (squares). Catalysis is in trans, i.e., the MuA subunit bound at R1 engages the L1 terminus and vice versa ( 9 ). This complex is not stable, and MuA has not tetramerized (indicated by a separation of the squares). (B) After Mu end synapsis, the free energy of supercoiling in the FD domain is used to melt several base pairs around the Mu–FD junction, concomitant with tetramerization of MuA (indicated by contiguous squares) ( 9 ). Mismatched substrates will tolerate any nucleotide at the terminal 2 bp, but require T at the +1 position on the nontransferred strand for stable MuA assembly. (C) Mismatched termini can cleave adjacent to any nucleotide at the +1 position on the transferred strand. The cleaved complex is more stable, indicated by a shape change to hexagons. (D) ST can occur on precleaved substrates even from an abasic site. This complex is the most stable, indicated by a shape change to the ovals. Each stage of transition (A–D) exhibits specific metal ion requirements and is regulated by MuB. See text for details.

Mu transpososome structures assembled on oligonucleotide substrates. (A) Two views of the 3D reconstruction of images of a cleaved MuA tetramer bound to R1 and R2 ends, obtained by scanning transmission electron microscopy at cryotemperatures, combined with electron spectroscopic imaging of the DNA-phosphorus ( 61 ). Target DNA is modeled into the structure. Location of Mu ends (black tubes) and FD (gray tubes) is indicated. The image has been modified from the original in order to match the orientation of the X-ray image in B. (B) X-ray crystal structure of the Mu transpososome engaged with cleaved R1 and R2 Mu ends joined to target DNA ( 62 ). The MuA polypeptide in the crystal structure includes residues 77 to 605; it is missing the regulatory N (Iα)- and C (IIIβ)-terminal domains (see Fig. 3B ). Left, schematic illustrating positions of the various MuA domains and DNA segments. Catalytic sites are marked as tan/yellow stars. Right, ribbon drawing, with the scissile phosphate groups shown as yellow spheres. The figure is modified to indicate position of the FD. In the crystal structure, the BAN region in domain IIIα (see Fig. 3B ) of the R2-bound subunits (cyan and pink) makes contact with the FD near the Mu–FD junction; this region is associated with a nonspecific endonuclease activity ( 122 ). Images in A ( 61 ) and B ( 62 ) are adapted with permission from the Nature Publishing group.

Interaction of MuA binding sites during transpososome assembly, and topology of the Mu DNA synapse. (A) 1. On a supercoiled DNA containing the native arrangement of L, E, and R segments, MuA-mediated interactions between E and R (square) trap two supercoil nodes within an initial ER synapse. 2. L is recruited into the ER complex with assistance from HU, and contributes one more crossing with E. The two L–R crossings within LER are fluid. 3. LER transitions into a stable complex (hexagon) which traps five supercoil nodes: two between E and R, one between E and L, and two between L and R. In this stable complex, the DNA around the Mu termini is first melted and then cleaved (see Fig. 4B, C ). 4. The five-noded LER topology is maintained in the ST complex (oval), which is the most stable. (B) Contribution of the individual MuA-binding sites to the DNA topology. R–E interactions, particularly R1–O1, are essential in the initial stages of assembly, R2–E interactions are not required, and R3-E interactions contribute to the distal E–R crossing (black dot). Six MuA subunits (not shown) hold the five DNA crossings. The MuA tetramer retains two L–R and the proximal E–R DNA crossings (black dots with white circles). Of the two L–R crossings, the one between L1 and R1 is likely the one seen in the crystal structure ( Fig. 5B ). Placement of the second L–R crossing is arbitrary; see reference 72 for details. IHF binding and bending at E between O1 and O2 optimizes E interactions with L and R. HU binding and bending L between L1 and L2 delivers L1 to the ER complex ( 47 ). (C) Encounter and synapsis of Mu ends on supercoiled DNA. In the absence of E, the L and R ends can approach each other either by slithering to form a plectonemically interwrapped (IW) synapse or by random collision to form a random collision (RC) synapse; the presence of E channels synapsis toward the IW pathway ( 77 ). See text for details.

The central SGS site helps Mu prophage ends pair. (A) Plectonemically supercoiled domains of the E. coli nucleoid are shown carrying a copy of the Mu genome; L and R ends and the centrally located SGS are indicated. DNA gyrase binds at the SGS and initiates processive introduction of supercoils, leading to the extrusion of a novel nucleoid domain comprising the Mu genome in its entirety, aligning the L and R ends to promote transpososome (MuA) assembly. (B) A model for the structure of a Mu prophage and for Mu genome immunity. The model proposes that segregation of Mu into a separate domain, as shown in (A), is sealed by either the Mu transpososome assembled on the ends, or by nucleoid associated proteins (NAPs). Several NAPs are shown stabilizing this structure, hypothesized to promote the formation of MuB filaments. MuB, which itself has NAP-like properties, is proposed to provide immunity to self-integration. Fis and H-NS proteins may be expected to reside at the SGS and Mu ends because these proteins prefer A/T-rich regions. SMC proteins have been proposed to be involved in the creation of large topological loops by bridging two DNAs at the base of the stem of such loops. (A) Adapted from reference 81 , and reprinted with permission from John Wiley and Sons. (B) Taken from references 85 and 106 .

Model for MuB function in target capture and cis immunity. (A) The helical parameters of the MuB filament (represented as beads on a string) do not match those of B-form DNA. This results in a nucleoprotein complex with a symmetry mismatch. (B) Matching symmetry between MuB and DNA would require the DNA to be underwound and extended, which may occur at the boundary of the MuB filament with the help of MuA and possibly ATP hydrolysis. Deformed and bent DNA is a preferred target for transposition catalyzed by MuA. (C) A summary of interplay among MuA, MuB, and DNA during transposition. Upon ATP binding, MuB forms helical filaments on DNA. MuA bound to Mu DNA ends stimulates ATP hydrolysis by MuB and MuB dissociation from DNA, which generates MuB-free DNA regions. Reciprocally, MuB stimulates MuA to pair and nick Mu DNA ends at the junction with the flanking sequences. MuA and MuB together may induce the matching symmetry between MuB and DNA at the boundary of a MuB filament and thus DNA distortion, which leads to the target DNA capture and Mu transposition. Taken from reference 34 , reprinted with permission from the Proceedings of the National Academy of Sciences U S A.

Transition from transposition to replication or repair. The ST intermediate in the lytic versus infection phase differs primarily in the configuration of the FD (see Fig. 1 ). (A) The depicted order of events was established from in vitro experiments ( 107 , 113 ). Mu replication is known to be unidirectional, primarily initiating at the L end ( 8 ). (B) Repair events are deduced from in vivo experiments ( 63 , 120 ). Question marks signify that the order of these events is not as yet established. X is a hypothetical factor. The arrow from B to A indicates that infecting Mu can proceed directly to replication without FD removal, as seen in a domain III MuA mutant defective in FD removal ( 63 ). See text for details of both pathways.