Modulation of phage Mu repressor DNA binding and degradation by distinct determinants in its C-terminal domain

Rapid degradation of the bacteriophage Mu immunity repressor can be induced in trans by mutant, protease-hypersensitive repressors (Vir) with an altered C-terminal domain (CTD). Genetic and biochemical analysis established that distinct yet overlapping determinants in the wild-type repressor CTD modulate Vir-induced degradation by Escherichia coli ClpXP protease and DNA binding by the N-terminal DNA-binding domain (DBD). Although deletions of the repressor C-terminus resulted in both resistance to ClpXP protease and suppression of a temperature-sensitive DBD mutation (cts62), some cysteine-replacement mutations in the CTD elicited only one of the two phenotypes. Some CTD mutations prevented degradation induced by Vir and resulted in the loss of intrinsic ClpXP protease sensitivity, characteristic of wild-type repressor, and at least two mutant repressors protected Vir from proteolysis. One protease-resistant mutant became susceptible to Vir-induced degradation when it also contained the cts62 mutation, which weakens DNA binding but apparently facilitates conversion to a protease-sensitive conformation. Conversely, this CTD mutation was able to suppress temperature sensitivity of DNA binding by the cts62 repressor. The results suggest that determinants in the CTD not only provide a cryptic ClpX recognition motif but also direct CTD movement that exposes the motif and modulates DNA binding.     

DNA-binding interactions and conformational fluctuations of Tc3 transposase DNA binding domain examined with single molecule fluorescence spectroscopy

The fluorescent dye tetramethylrhodamine (TMR) was conjugated to a synthetic peptide containing the sequence-specific DNA binding domain of Tc3 transposase. Steady-state and single molecule fluorescence spectroscopy was used to investigate protein conformational fluctuations and the thermodynamics of binding interactions. Evidence is presented to show that the TMR-Tc3 conjugate exists in at least two conformational states. The most stable conformation is one in which the TMR fluorescence is quenched. Upon binding to DNA, the total fluorescence from TMR-Tc3 increases by three- to fourfold. Single molecule measurements of TMR-Tc3 bound to DNA shows that this complex also fluctuates between a fluorescent and quenched form. The fluorescent form of the conjugate is stabilized when bound to DNA, and this accounts for part of the increase in total fluorescence. In addition, the inherent photodynamics of the dye itself is also altered (e.g., fluorescent lifetime or triplet yield) in such a way that the total fluorescence from the conjugate bound to DNA is enhanced relative to the unbound form.     

A novel DNA binding and nuclease activity in domain III of Mu transposase: evidence for a catalytic region involved in donor cleavage.

The Mu A protein is a 75 kDa transposase organized into three structural domains. By severing the C-terminal region (domain III) from the remainder of the protein, we unmasked a novel non-specific DNA binding and nuclease activity in this region. Deletion analysis localized both activities to a 26 amino acid stretch (aa 575-600) which remarkably remained active in DNA binding and cleavage. The two activities were shown to be tightly linked by site-directed mutagenesis. To study the importance of these activities in the transposition process, an intact mutant transposase lacking the DNA binding and nuclease activity of domain III was constructed and purified. The mutant transposase was indistinguishable from wild-type Mu A in binding affinity for both the Mu ends and the enhancer, and in strand transfer activity when the cleavage step was bypassed. In contrast, the mutant transposase displayed defects in both synapsis and donor cleavage. Our results strongly suggest that the 26 amino acid region in domain III carries catalytic residues required for donor DNA cleavage by Mu A protein. Furthermore, our data suggest that an active site for donor cleavage activity in the Mu tetramer is assembled from domain II (metal ion binding) in one A monomer and domain III (DNA cleavage) in a separate A monomer. This proposal for active site assembly is in agreement with the recently proposed domain sharing model by Yang et al. (Yang, J.Y., Kim, K., Jayaram, M. and Harshey, R.M. [1995] EMBO J., 14, 2374-2384).     

The solution structure of the C-terminal domain of the Mu B transposition protein

Mu B is one of four proteins required for the strand transfer step of bacteriophage Mu DNA transposition and the only one where no high resolution structural data is available. Structural work on Mu B has been hampered primarily by solubility problems and its tendency to aggregate. We have overcome this problem by determination of the three-dimensional structure of the C-terminal domain of Mu B (B(223-312)) in 1.5 M NaCl using NMR spectroscopic methods. The structure of Mu B(223-312) comprises four helices (backbone r.m.s.d. 0.46 A) arranged in a loosely packed bundle and resembles that of the N-terminal region of the replication helicase, DnaB. This structural motif is likely to be involved in the inter-domainal regulation of ATPase activity for both Mu A and DnaB. The approach described here for structural determination in high salt may be generally applicable for proteins that do not crystallize and that are plagued by solubility problems at low ionic strength.     

Mutational analysis of the att DNA-binding domain of phage Mu transposase.

The transposase (A protein) of phage Mu encodes binding to two families of DNA sites, att sites located at the Mu ends and enhancer sites located internally. Separate subdomains in the N-terminal domain I of Mu A protein are known to be involved in recognition of the att and enhancer sites. We have delineated an approximately 135 aa region within domain I beta gamma that specifies binding to Mu att sites. This peptide was overexpressed and its properties compared with that of the larger domain I beta gamma as well as the intact Mu A protein. Extensive mutagenesis of residues around a putative helix-turn-helix DNA-binding motif within the I beta domain identified several mutants defective in DNA transposition in vivo. Of these, Mu A(K157Q) was completely defective in att DNA-binding. Mu A(F131S) and Mu A(R146N) had a lower affinity for att DNA and low levels of transposition in vitro. Our results indicate that residues in the gamma region are required for activity and that residues outside the beta gamma region must also influence discrimination between the multiple att sites.     

A subsequence-specific DNA-binding domain resides in the 13 kDa amino terminus of the bacteriophage Mu transposase protein

We have previously reported that the 13 kDa amino terminus of the 70 kDa bacteriophage D108 transposase protein (A gene product) contains a two-component, sequence-specific DNA-binding domain which specifically binds to the related bacteriophage Mu's right end (attR) in vitro. To extend these studies, we examined the ability of the 13 kDa amino terminus of the Mu transposase protein to bind specifically to Mu attR in crude extracts. Here we report that the Mu transposase protein also contains a Mu attR specific DNA-binding domain, located in a putative alpha-helix-turn-alpha-helix region, in the amino terminal 13 kDa portion of the 70 kDa transposase protein as part of a 23 kDa fusion protein with beta-lactamase. We purified for this attR-specific DNA-binding activity and ultimately obtained a single polypeptide of the predicted molecular weight for the A'--'bla fusion protein. We found that the pure protein bound to the Mu attR site in a different manner compared with the entire Mu transposase protein as determined by DNase I-footprinting. Our results may suggest the presence of a potential primordial DNA-binding site (5'-PuCGAAA-3') located several times within attR, at the ends of Mu and D108 DNA, and at the extremities of other prokaryotic class II elements that catalyze 5 base pair duplications at the site of element insertion. The dissection of the functional domains of the related phage Mu and D108 transposase proteins will provide clues to the mechanisms and evolution of DNA transposition as a mode of mobile genetic element propagation.     

Interactions of the transposase with the ends of Mu: formation of specific nucleoprotein structures and non-cooperative binding of the transposase to its binding sites.

Transposition of the E. coli bacteriophage Mu requires the phage encoded A and B proteins, the host protein HU and the host replication proteins. The ends of the genome of the phage, on which some of these proteins act, both contain three transposase (A) binding sites. The organization of these binding sites on each end, however, is different. Here we show, using DNase footprinting experiments with purified A protein, that mutant A binding sites, which affect transposition, have decreased affinity for the transposase. Furthermore the transposase binds non-cooperatively to all A binding sites both in the left and right end of Mu. Electron microscopic studies show that the A protein forms specific nucleoprotein structures upon binding to the ends of Mu. The A and B proteins interact with the ends of Mu to generate larger structures than with the A protein alone.     

A bifunctional DNA binding region in Tn5 transposase

Tn5 transposition is a complicated process that requires the formation of a highly ordered protein-DNA structure, a synaptic complex, to catalyse the movement of a sequence of DNA (transposon) into a target DNA. Much is known about the structure of the synaptic complex and the positioning of protein-DNA contacts, although many protein-DNA contacts remain largely unstudied. In particular, there is little evidence for the positioning of donor DNA and target DNA. In this communication, we describe the isolation and analysis of mutant transposases that have, for the first time, provided genetic and biochemical evidence for the stage-specific positioning of both donor and target DNAs within the synaptic complex. Furthermore, we have provided evidence that some of the amino acids that contact donor DNA also contact target DNA, and therefore suggest that these amino acids help define a bifunctional DNA binding region responsible for these two transposase-DNA binding events.     

Tc1 transposase of Caenorhabditis elegans is an endonuclease with a bipartite DNA binding domain.

The Tc1 transposon of Caenorhabditis elegans is a member of the Tc1/mariner family of mobile elements. These elements have inverted terminal repeats that flank a single transposase gene. Here we show that Tc1 transposase, Tc1A, has a bipartite DNA binding domain related to the paired domain of mammalian and Drosophila genes. Both the DNA binding domain of Tc1A and the DNA binding site in the inverted repeat of Tc1 can be divided into two subdomains. Methylation interference studies demonstrate adjacent minor and major groove contacts at the inner part of the binding site by the N-terminal 68 amino acids of the DNA binding domain. In addition, Tc1A amino acids 69-142 are essential for major groove contacts at the outer part of the binding site. Recombinant Tc1A is found to be able to introduce a single strand nick at the 5' end of the transposon in vitro. Furthermore, Tc1A can mediate a phosphoryl transfer reaction. A mutation in a DDE motif abolishes both endonucleolytic and phosphoryl transfer activities, suggesting that Tc1A carries a catalytic core common to retroviral integrases and IS transposases.     

Efficient Mu transposition requires interaction of transposase with a DNA sequence at the Mu operator: implications for regulation

Phage Mu transposition is initiated by the Mu DNA strand-transfer reaction, which generates a branched DNA structure that acts as a transposition intermediate. A critical step in this reaction is formation of a special synaptic DNA-protein complex called a plectosome. We find that formation of this complex involves, in addition to a pair of Mu end sequences, a third cis-acting sequence element, the internal activation sequence (IAS). The IAS is specifically recognized by the N-terminal domain of Mu transposase (MuA protein). Neither the N-terminal domain of MuA protein nor the IAS is required for later reaction steps. The IAS overlaps with the sequences to which Mu repressor protein binds in the Mu operator region; the Mu repressor directly inhibits the Mu DNA strand-transfer reaction by interfering with the interaction between MuA protein and the IAS, providing an additional mode of regulation by the repressor.     

Characterization of amber mutations in bacteriophage Mu transposase: a functional analysis of the protein

We have characterized a series of amber mutations in the A gene of bacteriophage Mu encoding the phage transposase. We tested different activities of these mutant proteins either in a sup0 strain or in different sup bacteria. In conjunction with the results described in the accompanying paper by Bétermier et al. (1989) we find that the C-terminus of the protein is not absolutely essential for global transposase function, but is essential for phage growth. Specific binding to Mu ends is defined by a more central domain. Our results also reinforce the previous findings (Bétermier et al., 1987) that more than one protein may be specified by the A gene.     

Two mutations of basic residues within the N-terminus of HMG-1 B domain with different effects on DNA supercoiling and binding to bent DNA

High mobility group (HMG) 1 protein and its two homologous DNA-binding domains, A and B ("HMG-boxes"), can bend and supercoil DNA in the presence of topoisomerase I, as well as recognize differently bent and distorted DNA structures, including four-way DNA junctions, supercoiled DNA and DNA modified with anticancer drug cisplatin. Here we show that the lysine-rich part of the linker region between A and B domains of HMG-1, the (85)TKKKFKD(91) sequence that is attached to the N-terminus of the B domain within HMG-1, is a prerequisite for a preferential binding of the B domain to supercoiled DNA. The above sequence is also essential for a high-affinity binding of the B domain to DNA containing a site-specific major 1,2-d(GpG) intrastrand DNA adduct of cisplatin. Mutation of Arg(97), but not Lys(90) [Lys(90) forms a specific cross-link with platinum(II) in major groove of cisplatin-modified DNA; Kane, S. A., and Lippard, S. J. (1996) Biochemistry 35, 2180--2188], to alanine significantly (>40-fold) reduces affinity of the B domain to cisplatin-modified DNA, inhibits the ability of the B domain to bend (ligase-mediated circularization) or supercoil DNA, and results in a loss of the preferential binding of the B domain to supercoiled DNA without affecting the structural-specificity of the HMG-box for four-way DNA junctions. Some of the reported activities of the B domain are enhanced when the B domain is covalently linked to the A domain. We propose that binding of the A/B linker region within the major DNA groove helps the two HMG-1 domains to anchor to the minor DNA groove to facilitate their DNA binding and other activities.     

The ITR binding domain of the Mariner Mos-1 transposase

Mariner-like elements are widespread eukaryotic transposons, but Mos-1 is the only natural element that is known to be active. Little is known about the biochemistry of mariner transposition. The first step in the process is the binding of the transposase to the 5' and 3' inverted terminal repeats (ITRs) of the element. Using the 3' ITR of the element, we have determined the binding properties of a recombinant Mos-1 transposase produced in bacteria, and we have used deletion derivatives to localize the minimal ITR binding domain between amino acids 1 and 141. Its features and structure indicate that it differs from the ITR binding domain of the transposase encoded by Tc1-related elements.     

Stimulation of the Mu DNA strand cleavage and intramolecular strand transfer reactions by the Mu B protein is independent of stable binding of the Mu B protein to DNA.

Interactions between the Mu A and Mu B proteins are important in the early steps of the in vitro transposition of a mini-Mu plasmid. We have examined these interactions by assaying Mu B stimulation of Mu A-mediated strand cleavage and strand transfer reactions. We have previously shown that in the presence of ATP the Mu B protein can stimulate the Mu A-directed cleavage reaction of mini-Mu plasmids carrying a terminal base pair mutation (Surette, M.G., Harkness, T., and Chaconas, G. (1991) J. Biol. Chem. 266, 3118-3124). Here we demonstrate that in the absence of a non-Mu DNA target molecule the Mu B protein stimulates intramolecular integration of a mini-Mu in an ATP-dependent fashion. Furthermore, modification of the Mu B protein with N-ethylmaleimide severely compromises the ability of B to form a stable complex with DNA; however, the modified protein stimulates the strand cleavage and intramolecular strand transfer reactions as efficiently as the untreated protein. These results indicate that the Mu B protein is capable of stimulating the Mu A protein through direct interaction in the absence of stable Mu B-DNA complex formation. Our results increase the spectrum of Mu B protein activities and uncouple the stimulatory properties of the Mu B protein from stable DNA binding but not the ATP cofactor requirement.     

A consensus motif in the RFX DNA binding domain and binding domain mutants with altered specificity.

The RFX DNA binding domain is a novel motif that has been conserved in a growing number of dimeric DNA-binding proteins, having diverse regulatory functions, in eukaryotic organisms ranging from yeasts to humans. To characterize this novel motif, we have performed a detailed dissection of the site-specific DNA binding activity of RFX1, a prototypical member of the RFX family. First, we have performed a site selection procedure to define the consensus binding site of RFX1. Second, we have developed a new mutagenesis-selection procedure to derive a precise consensus motif, and to test the accuracy of a secondary structure prediction, for the RFX domain. Third, a modification of this procedure has allowed us to isolate altered-specificity RFX1 mutants. These results should facilitate the identification both of additional candidate genes controlled by RFX1 and of new members of the RFX family. Moreover, the altered-specificity RFX1 mutants represent valuable tools that will permit the function of RFX1 to be analyzed in vivo without interference from the ubiquitously expressed endogenous protein. Finally, the simplicity, efficiency, and versatility of the selection procedure we have developed make it of general value for the determination of consensus motifs, and for the isolation of mutants exhibiting altered functional properties, for large protein domains involved in protein-DNA as well as protein-protein interactions.     

Cloning of the A gene of bacteriophage Mu and purification of its product, the Mu transposase

The bacteriophage Mu transposase (the Mu A gene product), which is absolutely required for both integration of Mu and replicative transposition during the lytic cycle, has been overproduced by cloning the gene on a plasmid under the control of the phage lambda PL promoter. The protein has been purified to near homogeneity from the lysate of heat-induced cells of a strain carrying the plasmid. The purified protein is active as judged by its ability to complement Mu A- cell extracts for supporting Mu transposition in a cell-free reaction.