Versatile action of Escherichia coli ClpXP as protease or molecular chaperone for bacteriophage Mu transposition

The molecular chaperone ClpX of Escherichia coli plays two distinct functions for bacteriophage Mu DNA replication by transposition. As specificity component of a chaperone-linked protease, it recognizes the Mu immunity repressor for degradation by the peptidase component ClpP, thus derepressing Mu transposition functions. After strand exchange has been promoted by MuA transposase, ClpX alone can alter the conformation of the transpososome (the complex of MuA with Mu ends), and the remodeled MuA promotes transition to replisome assembly. Although ClpXP can degrade MuA, the presence of both ClpP and ClpX in the reconstituted transposition system did not destroy MuA essential for initiation of DNA replication by specific host replication enzymes. Levels of ClpXP needed to overcome inhibition by the repressor did not prevent MuA from promoting strand transfer, and ClpP stimulated alteration of the transpososome by ClpX. Apparently intact MuA was still present in the resulting transpososome, promoting initiation of Mu DNA replication by specific replication enzymes. The results indicate that ClpXP can discriminate repressor and MuA in the transpososome as substrates of the protease or the molecular chaperone alone, degrading repressor while remodeling MuA for its next critical function.     

The same two monomers within a MuA tetramer provide the DDE domains for the strand cleavage and strand transfer steps of transposition

The chemistry of Mu transposition is executed within a tetrameric form of the Mu transposase (MuA protein). A triad of DDE (Asp, Asp35Glu motif) residues in the central domain of MuA (DDE domain) is essential for both the strand cleavage and strand transfer steps of transposition. Previous studies had suggested that complete Mu transposition requires all four subunits in the MuA tetramer to carry an active DDE domain. Using a mixture of MuA proteins with either wild-type or altered att-DNA binding specificities, we have now designed specific arrangements of MuA subunits carrying the DDE domain. From analysis of the abilities of oriented tetramers to carry out DNA cleavage and strand transfer from supercoiled DNA, a new picture of the disposition of DNA and protein partners during transposition has emerged. For DNA cleavage, two subunits of MuA located at attL1 and attR1 (sites that undergo cleavage) provide DDE residues in trans. The same two subunits contribute DDE residues for strand transfer, also in trans. Thus, only two active DDE+ monomers within the tetramer carry out complete Mu transposition. We also show that when the attR1-R2 arrangement used on supercoiled substrates is tested for cleavage on linear substrates, alternative chemically competent DNA-protein associations are produced, wherein the functional DDE subunits are positioned at R2 rather than at R1.     

The phiX174-type primosome promotes replisome assembly at the site of recombination in bacteriophage Mu transposition

Initiation of Escherichia coli DNA synthesis primed by homologous recombination is believed to require the phiX174-type primosome, a mobile priming apparatus assembled without the initiator protein DnaA. We show that this primosome plays an essential role in bacteriophage Mu DNA replication by transposition. Upon promoting transfer of Mu ends to target DNA, the Mu transpososome undergoes transition to a pre-replisome that permits initiation of DNA synthesis only in the presence of primosome assembly proteins PriA, DnaT, DnaB and DnaC. These assembly proteins promote the engagement of primase and DNA polymerase III holoenzyme, initiating semi-discontinuous replication preferentially at the Mu left end. The results indicate that these proteins play a crucial role in promoting replisome assembly on a recombination intermediate.     

The Rep52 gene product of adeno-associated virus is a DNA helicase with 3'-to-5' polarity

The rep gene of adeno-associated virus type 2 encodes four overlapping proteins from two separate promoters, termed P5 and P19. The P5-promoted Rep proteins, Rep78 and Rep68, are essential for viral DNA replication, and a wealth of data concerning the biochemical activities of these proteins has been reported. In contrast, data concerning the biochemical functions of the P19-promoted Rep proteins, Rep52 and Rep40, are lacking. Here, we describe enzymatic activities associated with a bacterially expressed maltose-binding protein (MBP)-Rep52 fusion protein. Purified MBP-Rep52 possesses 3'-to-5' DNA helicase activity that is strictly dependent upon the presence of nucleoside triphosphate and divalent cation cofactors. In addition, MBP-Rep52 demonstrates a constitutive ATPase activity that is active in the absence of DNA effector molecules. An MBP-Rep52 chimera bearing a lysine-to-histidine substitution at position 116 (K116H) within a consensus helicase- and ATPase-associated motif (motif I or Walker A site) was deficient for both DNA helicase and ATPase activities. In contrast to a Rep78 A-site mutant protein bearing a corresponding amino acid substitution at position 340 (K340H), the MBP-Rep52 A-site mutant protein failed to exhibit a trans-dominant negative effect when it was mixed with wild-type MBP-Rep52 or MBP-Rep78 in vitro. This lack of trans dominance, coupled with the results of coimmunoprecipitation and gel filtration chromatography experiments reported here, suggests that the ability of Rep52 to engage in multimeric interactions may differ from that of Rep78 or -68.     

Nucleotidase activities of the 26 S proteasome and its regulatory complex

The 26 S proteasome can be assembled from the multicatalytic protease (or 20 S proteasome) and a large multisubunit regulatory complex in an ATP-dependent reaction. The 26 S proteasome and its regulatory complex were purified from rabbit reticulocytes for characterization of their nucleotidase properties. Both particles hydrolyze ATP, CTP, GTP, and UTP to the corresponding nucleoside diphosphate and inorganic phosphate. The Km values for hydrolysis of specific nucleotides by the 26 S proteasome are 15 microM for ATP and CTP, 50 microM for GTP, and 100 microM for UTP; Km values for nucleotide hydrolysis by the regulatory complex are 2-4-fold higher for each nucleotide. Several ATPase inhibitors (erythro-9-[3-(2-hydroxynonyl)]adenine, oligomycin, ouabain, and thapsigargin) had no effect on ATP hydrolysis by either complex whereas known inhibitors of proteolysis by the 26 S enzyme (hemin, N-ethylmaleimide (NEM), and vanadate) significantly reduced ATP hydrolysis by both particles. Hydrolysis of all nucleotides was inhibited by 5 mM NEM but only GTP and UTP hydrolysis was significantly reduced at 50 microM NEM. The 15 microM Km for ATP hydrolysis by the 26 S proteasome is virtually identical to the observed Km of 12 microM ATP for Ub-conjugate degradation. Although nucleotide hydrolysis is required for protein degradation by the 26 S proteasome, nucleotide hydrolysis and peptide bond cleavage are not strictly coupled. Substrate specificity constants (kcat/Km) are similar for hydrolysis of each nucleotide, yet GTP and UTP barely supported Ub-conjugate degradation. Further evidence that nucleotide hydrolysis is not coupled to peptide bond cleavage was obtained using N-acetyl-leucyl-leucyl-norleucinal (LLnL). This compound inhibited peptide hydrolysis by the multicatalytic protease and Ub-conjugate degradation by the 26 S proteasome, but it had little effect on ATP or UTP hydrolysis by the 26 S enzyme.     

An essential protease involved in bacterial cell-cycle control

Proteolytic inactivation of key regulatory proteins is essential in eukaryotic cell-cycle control. We have identified a protease in the eubacterium Caulobacter crescentus that is indispensable for viability and cell-cycle progression, indicating that proteolysis is also involved in controlling the bacterial cell cycle. Mutants of Caulobacter that lack the ATP-dependent serine protease ClpXP are arrested in the cell cycle before the initiation of chromosome replication and are blocked in the cell division process. ClpXP is composed of two types of polypeptides, the ClpX ATPase and the ClpP peptidase. Site-directed mutagenesis of the catalytically active serine residue of ClpP confirmed that the proteolytic activity of ClpXP is essential. Analysis of mutants lacking ClpX or ClpP revealed that both proteins are required in vivo for the cell-cycle-dependent degradation of the regulatory protein CtrA. CtrA is a member of the response regulator family of two-component signal transduction systems and controls multiple cell-cycle processes in Caulobacter. In particular, CtrA negatively controls DNA replication and our findings suggest that specific degradation of the CtrA protein by the ClpXP protease contributes to G1-to-S transition in this organism.     

DNA binding by the KP repressor protein inhibits P-element transposase activity in vitro

P elements are a family of mobile DNA elements found in Drosophila. P-element transposition is tightly regulated, and P-element-encoded repressor proteins are responsible for inhibiting transposition in vivo. To investigate the molecular mechanisms by which one of these repressors, the KP protein, inhibits transposition, a variety of mutant KP proteins were prepared and tested for their biochemical activities. The repressor activities of the wild-type and mutant KP proteins were tested in vitro using several different assays for P-element transposase activity. These studies indicate that the site-specific DNA-binding activity of the KP protein is essential for repressing transposase activity. The DNA-binding domain of the KP repressor protein is also shared with the transposase protein and resides in the N-terminal 88 amino acids. Within this region, there is a C2HC putative metal-binding motif that is required for site-specific DNA binding. In vitro the KP protein inhibits transposition by competing with the transposase enzyme for DNA-binding sites near the P-element termini.     

A new set of Mu DNA transposition intermediates: alternate pathways of target capture preceding strand transfer

Mu DNA transposition occurs within the context of higher order nucleoprotein structures or transpososomes. We describe a new set of transpososomes in which Mu B-bound target DNA interacts non-covalently with previously characterized intermediates prior to the actual strand transfer. This interaction can occur at several points along the reaction pathway: with the LER, the Type 0 or the Type 1 complexes. The formation of these target capture complexes, which rapidly undergo the strand transfer chemistry, is the rate-limiting step in the overall reaction. These complexes provide alternate pathways to strand transfer, thereby maximizing transposition potential. This versatility is in contrast to other characterized transposons, which normally capture target DNA only at a single point in their respective reaction pathways.     

An ecto-nucleotide pyrophosphatase is one of the main enzymes involved in the extracellular metabolism of ATP in rat C6 glioma

The presence of a nucleotide pyrophosphatase (EC 3.6.1.9) on the plasma membrane of rat C6 glioma has been demonstrated by analysis of the hydrolysis of ATP labeled in the base and in the alpha- and gamma-phosphates. The enzyme degraded ATP into AMP and PPi and, depending on the ATP concentration, accounted for approximately 50-75% of the extracellular degradation of ATP. The association of the enzyme with the plasma membrane was confirmed by ATP hydrolysis in the presence of a varying concentration of pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), a membrane-impermeable inhibitor of the enzyme. PPADS concentration above 20 microM abolished the degradation of ATP into AMP and PPi. The nucleotide pyrophosphatase has an alkaline pH optimum and a Km for ATP of 17 +/- 5 microM. The enzyme has a broad substrate specificity and hydrolyzes nucleoside triphosphates, nucleoside diphosphates, dinucleoside polyphosphates, and nucleoside monophosphate esters but is inhibited by nucleoside monophosphates, adenosine 3',5'-bisphosphate, and PPADS. The substrate specificity characterizes the enzyme as a nucleotide pyrophosphatase/phosphodiesterase I (PD-I). Immunoblotting and autoadenylylation identified the enzyme as a plasma cell differentiation antigen-related protein. Hydrolysis of ATP terminates the autophosphorylation of a nucleoside diphosphate kinase (NDPK/nm23) detected in the conditioned medium of C6 cultures. A function of the pyrophosphatase/PD-I and NDPK in the purinergic and pyrimidinergic signal transduction in C6 is discussed.     

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).     

A two-site mechanism for ATP hydrolysis by the asymmetric Rep dimer P2S as revealed by site-specific inhibition with ADP-A1F4

The Escherichia coli Rep helicase is a dimeric motor protein that catalyzes the transient unwinding of duplex DNA to form single-stranded (ss) DNA using energy derived from the binding and hydrolysis of ATP. In an effort to understand this mechanism of energy transduction, we have used pre-steady-state methods to study the kinetics of ATP binding and hydrolysis by an important intermediate in the DNA unwinding reaction--the asymmetric Rep dimer state, P2S, where ss DNA [dT(pT)15] is bound to only one subunit of the Rep dimer. To differentiate between the two potential ATPase active sites inherent in the dimer, we constructed dimers with one subunit covalently cross-linked to ss DNA and where one or the other of the ATPase sites was selectively complexed to the tightly bound transition state analog ADP-A1F4. We found that when ADP-A1F4 is bound to the Rep subunit in trans from the subunit bound to ss DNA, steady-state ATPase activity of 18 s(-1) per dimer (equivalent to wild-type P2S) was recovered. However, when the ADP-A1F4 and ss DNA are both bound to the same subunit (cis), then a titratable burst of ATP hydrolysis is observed corresponding to a single turnover of ATP. Rapid chemical quenched-flow techniques were used to resolve the following minimal mechanism for ATP hydrolysis by the unligated Rep subunit of the cis dimer: E + ATP <==> E-ATP <==> E'-ATP <==> E'-ADP-Pi <==> E-ADP-Pi <==> E-ADP + Pi <==> E + ADP + Pi, with K1 = (2.0 +/- 0.85) x 10(5) M(-1), k2 = 22 +/- 3.5 s(-1), k(-2) < 0.12 s(-1), K3 = 4.0 +/- 0.4 (k3 > 200 s(-1)), k4 = 1.2 +/- 0.14 s(-1), k(-4) < 1.2 s(-1), K5 = 1.0 +/- 0.2 mM, and K6 = 80 +/- 8 microM. A salient feature of this mechanism is the presence of a kinetically trapped long-lived tight nucleotide binding state, E'-ADP-Pi. In the context of our "subunit switching" model for Rep dimer translocation during processive DNA unwinding [Bjornson, K. B., Wong, I., & Lohman, T. M. (1996) J. Mol. Biol. 263, 411-422], this state may serve an energy storage function, allowing the energy from the binding and hydrolysis of ATP to be harnessed and held in reserve for DNA unwinding.     

Mutational analysis of the Mu transposase. Contributions of two distinct regions of domain II to recombination

Mu transposase is a member of a protein family that includes many transposases and the retroviral integrases. These recombinases catalyze the DNA cleavage and joining reactions essential for transpositional recombination. Here we demonstrate that, consistent with structural predictions, aspartate 336 of Mu transposase is required for catalysis of both DNA cleavage and DNA joining. This residue, although located 55 rather than 35 residues NH2-terminal of the essential glutamate, is undoubtedly the analog of the second aspartate of the Asp-Asp-35-Glu motif found in other family members. The core domain of Mu transposase consists of two subdomains: the NH2-terminal subdomain (IIA) contains the conserved Asp-Asp-Glu motif residues, whereas the smaller COOH-terminal subdomain (IIB) contains a large positively charged region exposed on its surface. To probe the function of domain IIB, we constructed mutant proteins carrying deletion or substitution mutations within this region. The activity of the deletion proteins revealed that domains IIA and IIB can be provided by different subunits in the transposase tetramer. Substitution mutations at two pairs of exposed lysine residues within the positively charged surface of domain IIB render transposase defective in transposition at a reaction step after DNA cleavage but prior to DNA joining. The severity of this defect depends on the structure of the DNA flanking the cleavage site. Thus, these data suggest that domain IIB is involved in manipulating the DNA near the cleavage site and that this function is important during the transition between the DNA cleavage and the DNA joining steps of recombination.     

The effect of nicotine on DNA repair in adult myocytes

Recent findings have demonstrated that terminally differentiated adult ventricular myocytes are capable of repairing DNA that has been damaged by exposure to oxygen free radicals. Despite the potential importance of DNA repair in cells that may survive many decades after injury, little is known about the mechanisms or regulation of repair. Since tobacco use has a well-defined role in the epidemiology and pathophysiology of heart disease, we tested the effects of nicotine on repair of free radical damaged plasmids by whole-cell protein extracts from adult myocytes. Exposure to a concentration of 25 microM nicotine increased incorporation of (32P)dCTP into damaged plasmids by 16%, and 50 or 100 microM nicotine increased incorporation by 32%. Nicotine did not alter the rate or amount of poly (ADP-ribose) on the major protein acceptor of molecular weight 113-116 kDa. Inhibition of DNA polymerase activity with pyridoxal 5'-phosphate revealed greater plasmid degradation in the presence of nicotine. We conclude that nicotine enhances DNA degradation and the increased repair is a consequence of this greater degradation.     

Altering the DNA-binding specificity of Mu transposase in vitro

We describe the isolation of a variant of Mu transposase (MuA protein) which can recognize altered att sites at the ends of Mu DNA. No prior knowledge of the structure of the DNA binding domain or its mode of interaction with att DNA was necessary to obtain this variant. Protein secondary structure programs initially helped target mutations to predicted helical regions within a subdomain of MuA demonstrated to harbor att DNA binding activity. Of the 54 mutant positions examined, only two showed decreased affinity for att DNA, while eight others affected assembly of the Mu transpososome. A variant impaired in DNA binding [MuA(R146V)], and predicted to be in the recognition helix of an HTH motif, was challenged with altered att sites created from degenerate oligonucleotides to select for novel DNA binding specificity. DNA sequences bound to MuA(R146V) were detected by gel-retardation, and following several steps of PCR amplification/enrichment, were identified by cloning and sequencing. The strategy allowed recovery of an altered att site for which MuA(R146V) showed higher affinity than for the wild-type site, although this site was bound by wild-type MuA as well. The altered association between MuA(R146V) and an altered att site target was competent in transposition. We discuss the strengths and limitations of this methodology, which has applications in dissecting the functional role of specific protein-DNA associations.     

DNA immunization: ubiquitination of a viral protein enhances cytotoxic T-lymphocyte induction and antiviral protection but abrogates antibody induction

DNA immunization can induce cytotoxic T lymphocytes (CTL), antibodies, and protection against microbial challenge. The underlying mechanisms remain obscure and must be understood to permit rational manipulation and optimization of the technique. We set out to enhance the intracellular degradation of a viral antigen, with the intent of improving antigen entry into, and presentation by, the class I major histocompatibility complex pathway. We achieved this goal by cotranslational ubiquitination of a plasmid-encoded viral antigen, lymphocytic choriomeningitis virus (LCMV) nucleoprotein (NP). We show that native NP is very stable in cell culture, while the ubiquitinated product is so rapidly degraded that it is barely detectable. This rapid degradation leads to more efficient sensitization of target cells in an in vitro cytotoxicity assay, consistent with enhanced antigen presentation, and both degradation and target cell recognition are blocked by a proteasome inhibitor. We have used the plasmid for in vivo studies and find that, remarkably, ubiquitination leads to a complete abrogation of antibody responses, presumably because the encoded protein is so rapidly and completely degraded that insufficient antigen remains to interact appropriately with B cells. In contrast, in vivo CTL induction is improved by ubiquitination of NP. That CTL are induced at all by this rapidly degraded protein may shed light on the mechanism by which CTL are induced by DNA immunization; it has been suggested that CTL induction following intramuscular DNA injection results not from antigen presentation by cells taking up and expressing the DNA but rather from uptake of soluble protein by specialized antigen-presenting cells (APC). It appears to us unlikely that the ubiquitinated protein could function in this manner, since it is so rapidly degraded in vitro and fails to induce antibodies in vivo. Finally, the ubiquitinated protein confers markedly enhanced protection against LCMV challenge. Mice immunized with a plasmid encoding NP show approximately 100-fold reductions in virus titers compared to controls, while mice immunized with a plasmid encoding the ubiquitinated NP show reductions in virus load of at least 5 x 10(4)- to 5 x 10(5)-fold. This is by far the most effective DNA vaccine that we have yet designed. Ubiquitination therefore may improve DNA immunization, but caution is warranted, since immunity to many microbes depends on induction of good humoral immunity, and we show here that this may be prevented by ubiquitination of the encoded protein.     

An ATP-ADP switch in MuB controls progression of the Mu transposition pathway

MuB protein, an ATP-dependent DNA-binding protein, collaborates with Mu transposase to promote efficient transposition. MuB binds target DNA, delivers this target DNA segment to transposase and activates transposase's catalytic functions. Using ATP-bound, ADP-bound and ATPase-defective MuB proteins we investigated how nucleotide binding and hydrolysis control the activities of MuB protein, important for transposition. We found that both MuB-ADP and MuB-ATP stimulate transposase, whereas only MuB-ATP binds with high affinity to DNA. Four different ATPase-defective MuB mutants fail to activate the normal transposition pathway, further indicating that ATP plays critical regulatory roles during transposition. These mutant proteins fall into two classes: class I mutants are defective in target DNA binding, whereas class II mutants bind target DNA, deliver it to transposase, but fail to promote recombination with this DNA. Based on these studies, we propose that the switch from the ATP- to ADP-bound form allows MuB to release the target DNA while maintaining its stimulatory interaction with transposase. Thus, ATP-hydrolysis by MuB appears to function as a molecular switch controlling how target DNA is delivered to the core transposition machinery.     

Adnexal torsion: endoscopic management, case reports, and review

Nucleotide-binding proteins are often used as molecular switches to control the assembly or activity of macromolecular machines. Recent work has revealed that such molecular switches also regulate the spread of some mobile DNA elements. Bacteriophage Mu and the bacterial transposon Tn7 each use an ATP-dependent molecular switch to select a new site for insertion and to coordinate the assembly of the transposition machinery at that site. Strong parallels between these ATP-dependent transposition proteins and other well-characterized molecular switches, such as Ras and EF-Tu, have emerged.     

Megavoltage imaging with a large-area, flat-panel, amorphous silicon imager

Mutations in an N-terminal 70-amino acid domain of bacteriophage Mu's repressor cause temperature-sensitive DNA-binding activity. Surprisingly, amber mutations can conditionally correct the heat-sensitive defect in three mutant forms of the repressor gene, cts25 (D43-G), cts62 (R47-Q) and cts71 (M28-I), and in the appropriate bacterial host produce a heat-stable Sts phenotype (for survival of temperature shifts). Sts repressor mutants are heat sensitive when in supE or supF hosts and heat resistant when in Sup degrees hosts. Mutants with an Sts phenotype have amber mutations at one of three codons, Q179, Q187, or Q190. The Sts phenotype relates to the repressor size: in Sup degrees hosts sts repressors are shorter by seven, 10, or 18 amino acids compared to repressors in supE or supF hosts. The truncated form of the sts62-1 repressor, which lacks 18 residues (Q179-V196), binds Mu operator DNA more stably at 42 degrees in vitro compared to its full-length counterpart (cts62 repressor). In addition to influencing temperature sensitivity, the C-terminus appears to control the susceptibility to in vivo Clp proteolysis by influencing the multimeric structure of repressor.