Purification and characterization of the T4 bacteriophage uvsX protein

Gene uvsX of bacteriophage T4 encodes a 40,000-dalton protein that plays a key role in the major pathway for genetic recombination in T4-infected cells. Mutations at the uvsX locus lead to increased sensitivity to various DNA-damaging agents, reduced phage bursts, decreased genetic recombination, and early arrest of DNA synthesis. Like the Escherichia coli recA protein, the purified uvsX protein is a DNA-dependent ATPase that catalyzes pairing between homologous single- and double-stranded DNA molecules in vitro (Yonesaki, T., Ryo, Y., Minagawa, T., and Takahashi, H., (1985) Eur. J. Biochem. 148, 127-134). At physiological salt concentrations, the uvsX protein binds tightly and cooperatively to single-stranded DNA, covering about five nucleotides per protein monomer; at lower salt concentrations, a similar type of binding to double-stranded DNA is detected (Griffith, J., and Formosa, T., (1985) J. Biol. Chem. 260, 4484-4491). We show here that the ATPase activity of this protein is unusual in producing both ADP plus Pi and AMP plus PPi as products. Generating the fully active form of the ATPase is a cooperative process, apparently requiring that a protein monomer be bound to single-stranded DNA while surrounded by other ATP-bound monomers. The catalysis of homologous pairing by the uvsX protein is shown to be greatly stimulated by the presence of the T4 gene 32 protein, a helix-destablizing protein previously studied in this laboratory, and it requires continued ATP hydrolysis. We present a method that allows the purification of the uvsX protein to essential homogeneity. We also describe the complete purification of two proteins that bind to the uvsX protein: the T4 uvsY protein (16,000 daltons) and an E. coli host protein of 32,000 daltons whose gene is unknown. The host protein is likely to play a role in DNA metabolism, because it also binds to the T4 gene 32 protein and to DNA; the sequence of its amino-terminal 29 amino acids has been determined.     

Crystal structure of a prokaryotic replication initiator protein bound to DNA at 2.6 A resolution.

The initiator protein (RepE) of F factor, a plasmid involved in sexual conjugation in Escherichia coli, has dual functions during the initiation of DNA replication which are determined by whether it exists as a dimer or as a monomer. A RepE monomer functions as a replication initiator, but a RepE dimer functions as an autogenous repressor. We have solved the crystal structure of the RepE monomer bound to an iteron DNA sequence of the replication origin of plasmid F. The RepE monomer consists of topologically similar N- and C-terminal domains related to each other by internal pseudo 2-fold symmetry, despite the lack of amino acid similarities between the domains. Both domains bind to the two major grooves of the iteron (19 bp) with different binding affinities. The C-terminal domain plays the leading role in this binding, while the N-terminal domain has an additional role in RepE dimerization. The structure also suggests that superhelical DNA induced at the origin of plasmid F by four RepEs and one HU dimer has an essential role in the initiation of DNA replication.     

On the binding of tRNA to Escherichia coli RNA polymerase. Interactions between the core enzyme, DNA and tRNA

We have investigated the interplay between the binding of tRNA and DNA to core RNA polymerase. We show that the monomer core enzyme can bind stably to either DNA or tRNA, whereas the dimer core can fix both DNA and tRNA in a stable ternary complex. We have examined the kinetics of the exchange between DNA and tRNA bound to the core enzyme. DNA bound to monomer core can be rapidly displaced by tRNA without prior dissociation of the core from the DNA. Similarly tRNA bound to the core can be displaced by DNA without prior dissociation of the tRNA. We confirm the result of Hinkle and Chamberlin [J. Mol. Biol. 70, 157-185 (1972)] that, in contrast, the core enzyme must first dissociate from one DNA molecule before it can transfer to another DNA. As this dissociation is very slow we suggest that, in vivo, the tRNA can act as a 'porter' providing the core enzyme with a more kinetically favourable path to transfer from one DNA site to another.     

Control of nitric oxide synthase dimer assembly by a heme-NO-dependent mechanism

Homodimer formation is a key step that follows heme incorporation during assembly of an active inducible nitric oxide synthase (iNOS). In cells, heme incorporation into iNOS becomes limited due to interaction between self-generated NO and cellular heme [Albakri, Q., and Stuehr, D. J. (1996) J. Biol. Chem. 271, 5414-5421]. Here we investigated if NO can regulate at points downstream in the process by inhibiting dimerization of heme-containing iNOS monomer. Heme-containing monomers were generated by treating iNOS dimer or iNOS oxygenase domain dimer (iNOSoxy) with urea. Both monomers dimerized when incubated with Arg and 6R-tetrahydrobiopterin (H4B), as shown previously [Abu-Soud, H. M., Loftus, M., and Stuehr, D. J. (1995) Biochemistry 34, 11167-11175]. The NO-releasing drug S-nitrosyl-N-acetyl-D,L-penicillamine (SNAP; 0-0.5 mM) inhibited dimerization of iNOS monomer in a dose- and time-dependent manner, without causing heme release. SNAP-pretreated monomer also did not dimerize in response to H4B plus Arg. SNAP converted Arg- and H4B-free iNOS dimer into monomer that could not redimerize, but had no effect on iNOS dimer preincubated with Arg and H4B. Anaerobic spectral analysis showed that NO from SNAP bound to the ferric heme of iNOSoxy monomer or dimer. Adding imidazole as an alternative heme ligand prevented SNAP from inhibiting iNOS monomer dimerization. We conclude that NO and related species can block iNOS dimerization at points downstream from heme incorporation. The damage to heme-containing monomer results from a reaction with the protein and appears irreversible. Although dimeric structure alone does not protect, it does enable Arg and H4B to bind and protect. Inhibition appears mediated by NO coordinating to the ferric heme iron of the monomer.     

Hydrogen exchange studies of the Arc repressor: Evidence for a monomeric folding intermediate

The hydrogen exchange rates of the backbone amide and labile side-chain protons of the dimeric Arc repressor have been measured. For the slowly exchanging amides in the α-helical regions, these rates show a concentration dependence. To account for this dependence, the role of the monomer–dimer equilibrium was considered. Extrapolating the observed exchange rates to zero dimer concentration provides estimates of these rates in the monomer and shows that they are significantly retarded compared to those of an unfolded polypeptide. This suggests that the monomer is in a structured “molten globule” like state. In particular, the two helices of Arc retain a high degree of their secondary structure and it is proposed that the two amphiphilic helices are packed together with their hydrophobic faces. Evidence for a partially folded structure in the Arc monomer was reported earlier in two other studies [J. L. Silva, C. F. Silveira, A. Correia, Jr., and L. Pontes (1992) Journal of Molecular Biology, Vol. 223, pp. 545–555: X. Peng, J. Jonas, and J. L. Silva (1993) Proceedings of the National Academy of Science USA, Vol. 90, pp. 1776–1780]. By combining the results of these studies and ours, a folding pathway of the dimeric Arc repressor involving four different stages is proposed. Due to the low concentration of Arc repressor in the cell, the protein is present either as a free monomer or it is bound to DNA presumably as a tetramer. Therefore the folding pathway can be regarded as an integral part of the overall DNA binding process. © 1995 John Wiley & Sons, Inc.     

Characterization and modification of a monomeric mutant of the lactose repressor protein

A monomeric mutant lactose repressor protein (T-41), containing serine at position 282 in place of tyrosine [Schmitz, A., Schmeissner, U., Miller, J. H., & Lu, P. (1976) J. Biol. Chem. 251, 3359-3366], has been purified by a series of chromatographic and precipitation methods. The molecular weight of the mutant as determined by gel filtration was approximately 40,000. The inducer equilibrium binding constant for the mutant was comparable to that of the tetrameric wild-type repressor at pH 7.5, whereas operator DNA binding was not detectable. In contrast to wild-type repressor, equilibrium and kinetic rate constants for inducer binding to the monomer were largely independent of pH; thus, the quaternary structure of the wild-type repressor is required for the pH-associated effects on inducer binding. Although ultraviolet absorbance difference spectra indicated that inducer binding to T-41 protein elicited different changes in the environment of aromatic residues from those generated in wild-type repressor, the shift in the fluorescence emission maximum in response to inducer binding was similar for T-41 and wild-type repressors. Similarity in 1-anilinonaphthalene-8-sulfonic acid binding to monomer and tetramer suggests that this fluorophore does not bind at subunit interfaces. Modification of Cys-281 with methyl methanethiosulfonate was observed at low molar ratios of reagent per T-41 monomer (4-fold). This result is in contrast to data observed for tetrameric wild-type repressor which requires high molar ratios for this cysteine to react. We conclude that Cys-281, adjacent to the site of the T-41 mutation, is located on the surface of the monomer in this region crucial for subunit interaction.     

Spo0J regulates the oligomeric state of Soj to trigger its switch from an activator to an inhibitor of DNA replication initiation

Control of DNA replication initiation is essential for bacterial cells to co-ordinate the faithful replication and segregation of their genetic material. The Bacillus subtilis ATPase Soj is a dynamic protein that regulates DNA replication initiation by either inhibiting or activating the DNA replication initiator protein DnaA. Here we report that the key event which switches Soj regulatory activity is a transition in its oligomeric state from a monomer to an ATP-dependent homodimer capable of DNA binding. We show that the DNA binding activity of the Soj dimer is required both for activation of DNA replication initiation and for interaction with Spo0J. Finally, we demonstrate that Spo0J inhibits Soj dimerization by stimulating Soj ATPase activity. The data provide a molecular explanation for the dichotomous regulatory activities of Soj, as well as assigning unique Soj conformations to distinct cellular localization patterns. We discuss how the regulation of Soj ATPase activity by Spo0J could be utilized to control the initiation of DNA replication during the cell cycle.     

Characterization of the Escherichia coli F factor traY gene product and its binding sites.

The traY gene product (TraYp) from the Escherichia coli F factor has previously been purified and shown to bind a DNA fragment containing the F plasmid oriT region (E. E. Lahue and S. W. Matson, J. Bacteriol. 172:1385-1391, 1990). To determine the precise nucleotide sequence bound by TraYp, DNase I footprinting was performed. The TraYp-binding site is near, but not coincident with, the site that is nicked to initiate conjugative DNA transfer. In addition, a second TraYp binding site, which is coincident with the mRNA start site at the traYI promoter, is described. The Kd for each binding site was determined by a gel mobility shift assay. TraYp exhibits a fivefold higher affinity for the oriT binding site compared with the traYI promoter binding site. Hydrodynamic studies were performed to show that TraYp is a monomer in solution under the conditions used in DNA binding assays. Early genetic experiments implicated the traY gene product in the site- and strand-specific endonuclease activity that nicks at oriT (R. Everett and N. Willetts, J. Mol. Biol. 136:129-150, 1980; S. McIntire and N. Willetts, Mol. Gen. Genet. 178:165-172, 1980). As this activity has recently been ascribed to helicase I, it was of interest to see whether TraYp had any effect on this reaction. Addition of TraYp to nicking reactions catalyzed by helicase I showed no effect on the rate or efficiency of oriT nicking. Roles for TraYp in conjugative DNA transfer and a possible mode of binding to DNA are discussed.     

Characterization of a Tn5 pre-cleavage synaptic complex

Protein catalyzed DNA rearrangements typically require assembly of complex nucleoprotein structures. In transposition and integration reactions, these structures, termed synaptic complexes, are mandatory for catalysis. We characterize the Tn5 pre-cleavage synaptic complex, the simplest transposition complex described to date. We identified this complex by gel retardation assay using short, linear fragments and have shown that it contains a dimer of transposase, two DNA molecules, and is competent for DNA cleavage in the presence of Mg(2+). We also used hydroxyl radical footprinting and interference techniques to delineate the protein-DNA contacts made in the Tn5 synaptic and monomer complexes. All positions (except position 1) of the end sequence are contacted by transposase in the synaptic complex. We have determined that positions 2-5 of the end sequence are specifically required for synaptic complex formation as they are not required for monomer complex formation. In addition, in the synaptic complex, there is a strong, local distortion centered around position 1 which likely facilitates cleavage.     

Nuclear deoxyribonucleic acid polymerase. Further observations on the structure and properties of the enzyme from human KB cells.

At low ionic strength KB cell DNA polymerase N1 forms large aggregates of a size comparable to those of DNA polymerase C. However, in contrast to polymerase C, the polymerase N1 aggregate: (a) retains the distinctive features of the polymerase N1 monomer, specifically its relative insensitivity to salt and to p-hydroxymercuribenzoate, and its pI of 9.3; and (b) is quantitatively converted to the polymerase N1 monomer form at appropriate ionic strength. It is important to recognize that since both polymerase N1 and polymerase C undergo salt-dependent association-dissociation reactions, attempts to distinguish these clearly indedependent polymerase species on the basis of size criteria can be very misleading. This is particularly true in relatively impure enzyme fractions that are generally isolated from eukaryotic tissue sources in low ionic strength buffers. We had earlier reported (Wang, T. S.-F., Sedwick, W. D., and Korn, D. (1974) J. Biol. Chem. 249,841-850; Sedwick, W. D., Wang, T. S.-F., and Korn, D. (1972) J. Biol. Chem. 247,5026-5033; Sedwick, W. D., Wang, T. S.-F., and Korn, D. (1974) Methods Enzymol. 29, 89-102) that DNA polymerase N1 could not utilize homoribopolymer templates. We have re-examined this question with a modified and more stringent method of product assay, and we show here that a greater than or equal 95% homogeneous preparation of polymerase N1 can copy the primer-template (A)n-(dT)-/16 at about one-half the rate that it copies activated DNA under optimum incubation conditions.     

V(D)J recombination: evidence that a replicative mechanism is not required.

We examined a series of extrachromosomal DNA substrates for V(D)J recombination under replicating and nonreplicating conditions. Complete and partial replications were examined by monitoring the loss of prokaryote-specific adenine methylation at 14 to 22 MboI-DpnI restriction sites (GATC) on the substrates. Some of these sites are within 2 bases of the signal sequence ends. We found that neither coding joint nor signal joint formation requires substrate replication. After ruling out replication as a substrate requirement, we determined whether replication had any effect on the efficiency of V(D)J recombination. Quantitation of V(D)J recombination efficiency on nonreplicating substrates requires some method of monitoring the entry of substrate molecules into the cells. We devised such a method by monitoring DNA repair of substrates into which we had substituted deoxyuridine for 10 to 20% of the thymidine nucleotides in the DNA. The substrates which enter the lymphoid cells were repaired efficiently in vivo by the eukaryotic uracil DNA repair system. Upon plasmid harvest, we distinguished repaired (entered) from unrepaired (not entered) plasmids by cleaving unrepaired molecules with uracil DNA glycoylase and Escherichia coli endonuclease IV in vitro. This method of monitoring DNA entry does not appear to underestimate or overestimate the amount of DNA entry. By using this method, we found no significant quantitative effect of DNA replication on V(D)J recombination efficiency.     

DNA gyrase-catalyzed decatenation of multiply linked DNA dimers

One possible intermediate during the terminal stages of the replication of a closed circular DNA is a catenated DNA dimer of the two completed daughter molecules. The two monomer DNA rings in these DNA dimers can be linked as many as 20-30 times. In Escherichia coli, DNA gyrase could act on these catenated dimers to eliminate the linkages between the daughter duplexes, yielding the final monomer product. In this report, this reaction has been studied biochemically. The in vitro pBR322 DNA replication system (Minden, J., and Marians, K. J. (1985) J. Biol. Chem. 260, 9316-9325) was used to manufacture large amounts of multiply linked catenated DNA dimers for use as a substrate for DNA gyrase-catalyzed decatenation. Studies presented here demonstrate that this decatenation reaction is more efficient with supercoiled as opposed to relaxed DNA dimers, proceeds in a distributive fashion, is inhibited by moderate amounts of salt (80 mM KCl), and is stimulated by the E. coli protein HU.     

Plasmid recombination intermediates generated in a Saccharomyces cerevisiae cell-free recombination system.

We have developed an assay utilizing Saccharomyces cerevisiae cell extracts to catalyze recombination in vitro between homologous plasmids containing different mutant alleles of the tet gene. Electrophoretic analysis of product DNA indicated that a number of novel DNA species were formed during the reaction. These species migrated through agarose gels as distinct bands with decreased electrophoretic mobility compared with the substrate DNA. The DNA from each individual band was purified and shown to be enriched 5- to 100-fold for tetracycline-resistant recombinants by using a transformation assay. The structure of the DNA molecules present in these bands was determined by electron microscopy. Recombination between circular substrates appeared to involve the formation and processing of figure-eight molecules, while recombination between circular and linear substrates involved the formation of molecules in which a circular monomer had a monomer-length linear tail attached at a region of homology.     

The bacteriophage Mu N gene encodes the 64-kDa virion protein which is injected with, and circularizes, infecting Mu DNA

Upon infection of Escherichia coli with bacteriophage Mu, a 64-kDa protein is injected into the host cell along with the phage DNA. This protein is involved in circularizing the infecting Mu DNA (Harshey, R. M., and Bukhari, A. I. (1983) J. Mol. Biol. 167, 427-441; Puspurs, A. H., Trun, N. J., and Reeve, J. N. (1983) EMBO J. 2, 345-352). Its possible role in the integration of infecting Mu DNA and in the infection process remains to be established. To identify the source of this protein we have prepared antiserum to the protein purified from viral particles. We have shown that the antiserum is specific for the Mu N gene product. The antiserum has been used to immunologically screen a Mu DNA library cloned into an expression vector. Four clones have been shown to produce a protein of 64 kDa that is specifically bound by the antiserum. The only Mu gene common to all four clones is the N gene, as demonstrated by physical and genetic mapping. We have also demonstrated by peptide mapping that the cloned N gene product is identical to the 64-kDa protein found complexed with the injected Mu DNA.     

Orientation of actin monomer in the F-actin filament: radial coordinate of glutamine-41 and effect of myosin subfragment 1 binding on the monomer orientation

We have employed the method of radial distance measurements in order to orient the actin monomer in the F-actin filament. This method utilizes fluorescence resonance energy transfer measurements of the distance between two equivalent chemical points located on two different monomers. The interprobe distance obtained this way is used to compute the radial coordinate of the labeled amino acid [Taylor, D. L., Reidler, J., Spudich, J. A., & Stryer, L. (1981) J. Cell Biol. 89, 362-367]. Theoretical analysis has indicated that if radial coordinates of four points are determined and six intramolecular distances are known, one can, within symmetry limits, position the monomer about the filament axis. The radial distance of Gln-41 that had been enzymatically modified with dansyl, rhodamine, and fluorescein derivatives of cadaverine was found to be approximately 40-42 A. The determination of the radial distance of Cys-374 was accomplished by using monobromobimane and N-[[(iodoacetyl)amino]ethyl]-5- naphthylamine-1-sulfonate as donors and N-[4-[[4-(dimethylamino)phenyl]azo]phenyl]maleimide as acceptor; the results were consistent with a radial coordinate for this residue of 20-25 A. The effect of myosin subfragment 1 (S1) binding on the radial coordinates of (1) Gln-41, (2) Cys-374, and (3) the nucleotide binding site was also examined. S1 had a small effect on the radial coordinate of Gln-41, increasing it to 44-47 A. In the two remaining lases the change in the radial coordinate due to the S1 binding was negligible. This finding excludes certain models of the interaction between actin and S1 in which actin monomer rotates by a large angle when subfragment 1 binds to it.     

The herpes simplex virus processivity factor, UL42, binds DNA as a monomer

The processivity subunit of the herpes simplex virus DNA polymerase, UL42, is a monomer in solution. However, UL42 is structurally similar to sliding clamp processivity factors, such as PCNA, which encircle DNA as a multimeric ring. We used chemical crosslinking and electrophoretic mobility-shift assays to investigate whether UL42 oligomerizes upon DNA binding. UL42 did not form intermolecular crosslinks upon treatment with glutaraldehyde in the presence of DNA, whereas proteins that are known to be multimers in solution were successfully crosslinked by this treatment. This result suggests that UL42 does not form multimers on DNA. We next analyzed the composition of UL42:DNA complexes using electrophoretic mobility-shift assays. UL42 was mixed with a maltose-binding protein-UL42 fusion protein before being added to DNA. The patterns of electrophoretic mobility of the resultant protein:DNA complexes were those predicted if each isoform of UL42 binds to DNA as a monomer. From this result and the failure of UL42 to form crosslinks, we infer that UL42 binds DNA as a monomer.     

Microinjected oligonucleotides complementary to the alpha-sarcin loop of 28 S RNA abolish protein synthesis in Xenopus oocytes

The integrity of the alpha-sarcin loop in 28 S ribosomal RNA is critical during protein synthesis. The toxins alpha-sarcin, ricin, Shiga toxin, and Shiga-like toxin inhibit protein synthesis in oocytes by attacking specific nucleotides within this loop (Ackerman, E.J., Saxena, S. K., and Ulbrich, N. (1988) J. Biol. Chem. 263, 17076-17083; Saxena, S.K., O'Brien, A.D., and Ackerman, E.J. (1989) J. Biol. Chem. 264, 596-601). We injected Xenopus oocytes with deoxyoligonucleotides complementary to the 17-nucleotide alpha-sarcin loop of Xenopus 28 S rRNA. Only injected oligonucleotides fully covering the alpha-sarcin loop or slightly beyond inhibited oocyte protein synthesis. Shorter alpha-sarcin domain deoxyoligonucleotides complementary to the alpha-sarcin and ricin sites but not spanning the entire loop were less effective inhibitors of protein synthesis. The alpha-sarcin domain oligonucleotides covering the entire loop were more effective inhibitors of protein synthesis than injected cycloheximide at equivalent concentrations. Control oligonucleotides complementary to nine other regions of Xenopus 28 S rRNA as well as universal M13 DNA sequencing primers had no effect on oocyte protein synthesis. Oligonucleotides complementary to the highly conserved alpha-sarcin domain therefore represent an alternative to catalytic toxins for causing cell death and may prove effective in immunotherapy.     

Doubling of the cytoplasm volume improves the developmental competence of porcine oocytes injected with freeze-dried somatic cells

In the present study using pig cells, we examined the effect of the cryoprotectant trehalose on the DNA integrity of freeze-dried cells. We then investigated whether donor cell types and storage duration had impact on DNA integrity in freeze-dried cells or developmental competence of oocytes injected with freeze-dried somatic cells. We also examined whether double cytoplasm nuclear transfer (DCNT) would improve developmental competence of such oocytes. Furthermore, using a PCR-based method for sex identification, we determined whether the blastocysts obtained had actually been generated from the freeze-dried cells. It was found that, for a short storage duration at low temperature, trehalose had no beneficial effect on protection from DNA damage, and that donor cell type had no effect on the DNA integrity of freeze-dried somatic cells or the developmental competence of oocytes injected with them. We also confirmed that all of the blastocysts obtained following nuclear transfer were of freeze-dried somatic cell origin. Storage of freeze-dried somatic cells for up to 1 year at low temperature did not degrade DNA integrity in comparison with storage for 1 month, 1 week or 1 day. Following injection of freeze-dried cells, the proportion of oocytes that developed to blastocysts after storage for up to 1 year was similar to that after storage for 1 month, 1 week or 1 day. Moreover, DCNT significantly improved the developmental competence of oocytes treated in this way. In summary, using DCNT, we have demonstrated that freeze-dried porcine somatic cells subjected to long-term storage at 4 °C have nearly the same potential to develop to blastocysts as non-freeze-dried cells.