DNA repair by the cryptic endonuclease activity of Mu transposase

Abstract

Phage Mu transposes by two distinct pathways depending on the specific stage of its life cycle. A common strand transfer intermediate is resolved differentially in the two pathways. During lytic growth, the intermediate is resolved by replication of Mu initiated within the flanking target DNA; during integration of infecting Mu, it is resolved without replication, by removal and repair of DNA from a previous host that is still attached to the ends of the incoming Mu genome. We have discovered that the cryptic endonuclease activity reported for the isolated C-terminal domain of the transposase MuA [Wu Z, Chaconas G (1995) A novel DNA binding and nuclease activity in domain III of Mu transposase: Evidence for a catalytic region involved in donor cleavage. EMBO J 14:3835-3843], which is not observed in the full-length protein or in the assembled transpososome in vitro, is required in vivo for removal of the attached host DNA or "5'flap" after the infecting Mu genome has integrated into the E. coli chromosome. Efficient flap removal also requires the host protein ClpX, which is known to interact with the C-terminus of MuA to remodel the transpososome for replication. We hypothesize that ClpX constitutes part of a highly regulated mechanism that unmasks the cryptic nuclease activity of MuA specifically in the repair pathway.

Fig. 1.

Two outcomes of Mu DNA transposition. A common θ intermediate is resolved differentially during replicative vs nonreplicative Mu transposition.

Fig. 2.

Endonuclease, DNA-binding, and transposition assays. (A) Domain organization of MuA showing the various functions mapped to individual domains. Residues targeted for mutagenesis in the BAN domain are boxed and in gray letters; the multiple changes made in three mutants in this region are indicated underneath. (B) Endonuclease and DNA-binding activities of isolated domain III (His-tagged MuA residues 575–663) carrying indicated mutations in residues 575–579. We surmise that addition of SDS triggered the nuclease, accounting for presence of DNA in the shifted complex in WT DNA-binding (no-SDS) lane. OC and SC, open circular and supercoiled forms of pUC19. (C) Type II (strand transfer) and Type II-2 (ClpX-mediated disassembly) reactions using full-length MuA and its BAN variants. D, donor pSP104; T, target pUC19; θ, disassembled θ topoisomers.

Fig. 3.

Flap removal from integrated Mu following infection with MuA BAN domain mutants in vivo. (A) Experimental strategy for monitoring flap removal. (B) Wild-type host BU1384 was infected with phages carrying wild-type or indicated mutants in the MuA BAN region. Mu (B gene) or flap sequences (MuR-lacZ) were amplified from isolated genomic DNA at indicated times after infection. 50 ng of DNA was used in all reactions with 30 cycles for detecting Mu and 40 cycles for detecting flaps. C is a control where genomic DNA from uninfected cells was mixed with Mu DNA and isolated by PFGE, to gauge the extent of contamination of the genomic DNA band with free Mu in subsequent PCRs. DNA loading was standardized to a chromosomal marker tsr. N, no template control, M, input Mu. (C) As in B, except infection was in a himA host (BU1382). Sixty nanograms DNA (30 cycles) was used for detecting Mu and 75 ng (40 cycles) for detecting the flap. The results in each panel were verified in at least three independent repeats. (D) The flap DNA bands in panel C were quantified as described in Methods and normalized to the signal from a chromosomal gene marker tsr.

Fig. 4.

Flap removal is delayed in a clpX but not a priA mutant. (A) Posttransposition events leading to Mu replication. The transpososome has six MuA subunits, four of which are tightly associated and two more loosely held. ClpX disassembles the complex, exchanging MuA with a series of protein factors in preparation for replication. The Mu-host joints after transpososome disassembly by ClpX are drawn according to ref. . [Reproduced with permission from ref.  (Copyright 2007, Molecular Microbiology).] See text for details. (B) Wild-type Mu was used to infect wild-type, clpX or priA mutant hosts. Reaction conditions as in Fig. 3B. (C) The flap DNA bands in indicated infections were analyzed as in Fig. 3D.

Fig. 5.

Model for domain III contribution to cleavage of nontransferred strands. Two catalytic subunits positioned at Mu termini, act in trans to carry out successive cleavage and strand transfer. The transferred strand is engaged in the DDE active site (domain II), whereas the nontransferred strand is engaged in the BAN active site (domain III). Domain IIIs could be contributed by noncatalytic subunits (19), indicated by unconnected hinge regions. Arrowheads, specific cleavage by BAN endonuclease. Mu (Thin Lines), target (Dotted Lines), and flap (Thick Lines).