Hexamminecobalt(III)-induced condensation of calf thymus DNA: circular dichroism and hydration measurements

The interaction of hexamminecobalt(III), Co(NH₃)₆³⁺, with 160 and 3000–8000 bp length calf thymus DNA has been investigated by circular dichroism, acoustic and densimetric techniques. The acoustic titration curves of 160 bp DNA revealed three stages of interaction: (i) Co(NH₃)₆³⁺ binding up to the molar ratio [Co(NH₃)₆³⁺]/[P] = 0.25, prior to DNA condensation; (ii) a condensation process between [Co(NH₃)₆³⁺]/[P] = 0.25 and 0.30; and (iii) precipitation after [Co(NH₃)₆³⁺]/[P] = 0.3. In the case of 3000–8000 bp DNA only two processes were observed: (i) binding up to [Co(NH₃)₆³⁺]/[P] = 0.3; and (ii) precipitation after this point. In agreement with earlier observations, long DNA aggregates without changes in its B-form circular dichroism spectrum, while short DNA demonstrates a positive B→Ψ transition after [Co(NH₃)₆³⁺]/[P] = 0.25. From ultrasonic and densimetric measurements the effects of Co(NH₃)₆³⁺ binding on volume and compressibility have been obtained. The binding of Co(NH₃)₆³⁺ to both short and long DNA is characterized by similar changes in volume and compressibility calculated per mole Co(NH₃)₆³⁺: ΔV = 9 cm³ mol^–1 and Δκ = 33 × 10^–4 cm³ mol^–1 bar^–1. The positive sign of the parameters indicates dehydration, i.e. water release from Co(NH₃)₆³⁺ and the atomic groups of DNA. This extent of water displacement would be consistent with the formation of two direct, hydrogen bonded contacts between the cation and the phosphates of DNA.     

Hydration effects of Ni(2+) binding to synthetic polynucleotides with regularly alternating purine-pyrimidine sequences

High precision ultrasonic and densimetric techniques have been used to study the interaction of Ni²⁺ ions with right-handed poly[d(G-C)]·poly[d(G-C)], poly-[d(A-C)]·poly[d(G-T)] and poly[d(A-T)]·poly[d(A-T)] in 5 mM CsCl, 0.2 mM HEPES, pH 7.5 at 20°C. From these measurements the changes in the apparent molar volume and the apparent molar adiabatic compressibility due to the interaction have been obtained. The volume effects of the binding, calculated per mole of Ni²⁺ ions, range from 11.7 to 23.9 cm³ mol^–1 and the compressibility effects range from 19.3 × 10^–4 to 43.1 × 10^–4 cm³ mol^–1 bar^–1. These data are interpreted in terms of dehydration of the polynucleotides and Ni²⁺ ions, i.e. the release of water molecules from the hydration shells of the molecules. An increase in G+C content gives an increase in volume and compressibility effects, indicating a rise in the extent of dehydration. The dehydration effects of Ni²⁺ binding to poly[d(G-C)]·poly[d(G-C)] are approximately twice those of poly[d(A-T)]·poly[d(A-T)]. The volume and compressibility effects of Ni²⁺–EDTA complex formation have also been measured and used as a model system for quantitative estimation. These values revealed that Ni²⁺ ions can coordinate two atomic groups of poly[d(G-C)]·poly[d(G-C)], while in the case of the Ni²⁺–poly[d(A-T)]·poly[d(A-T)] complex volume and compressibility effects correspond to one direct or two indirect (through water) contacts.     

Reactions of protamine with the molecular chaperone DnaK

Molecular chaperones of the 70 kDa family mediate protein–protein interactions by selectively binding to partially unfolded segments of other proteins in an ATP-dependent activity cycle. Previous investigations of chaperone substrate selectivity have shown that chaperones have a propensity to bind to partially unfolded segments of polypeptides that contain bulky hydrophobic residues. However, recent investigations have shown that 70 kDa chaperones such as DnaK, which is expressed by Escherichia coli, also bind short basic peptides and even polycations. We report here that DnaK specifically binds to the polycation protamine when [protamine]/[DnaK] is near unity, whereas protamine induces the aggregations of DnaK when [protamine]/[DnaK] ≥ 20. Complexes between DnaK and protamine were detected using fluorescently labeled protamine (protamine*) in conjunction with high performance size exclusion chromatography. We found that: (i) an unlabeled peptide of known affinity for DnaK partially inhibited the formation of DnaK-protamine* complexes; (ii) Mg-ATP (and Mg-γ-S-ATP) significantly reduced the affinity of protamine* for DnaK; and (iii) the rate of DnaK-protamine* complex dissociation is highly temperature-sensitive, with apparent activation enthalpies (ΔH*) equal to 32 ± 4 and 28 ± 1 kcal mol⁻¹ in the absence of added nucleotide and in the presence of ADP, respectively. The results are consistent with the specific binding of protamine* at the (poly)peptide binding site of DnaK. A model is proposed to account for the protamine-induced aggregation of DnaK.     

Direct BRLF1 binding is required for cooperative BZLF1/BRLF1 activation of the Epstein – Barr virus early promoter, BMRF1

Disruption of Epstein – Barr virus (EBV) latency is mediated through the activation of the viral immediate-early proteins, BZLF1 (Z) and BRLF1 (R).i.; (Chevallier-Greco, A., et al., (1986) EMBO J., 5, 3243 – 9; Countryman, and Miller, G. (1985) Proc. Natl. Acad. Sci. USA, 82, 4085 – 4089). We have previously demonstrated that these proteins cooperatively activate the EBV early promoter BMRF1 in lymphoid cells but not in epithelial cells. Although cooperative transactivation by these proteins has been demonstrated with a number of EBV promoters, the mechanism of this interaction is not well understood. We now show that the cooperative activation of the BMRF1 promoter by Z-plus-R requires an intact R binding site and at least one functional Z response element (ZRE). Despite the presence of an R binding site, the BMRF1 promoter is only moderately responsive to R alone in either HeLa or Jurkat cells. Efficient activation of the BMRF1 promoter by Z alone in HeLa cells requires two ZREs (located at − 59 and − 106), whereas two additional Z binding sites (located at − 42 and − 170) contribute very little to Z-induced activation. In the absence of ZREs, Z acted as a repressor of R-induced transactivation. These observations, along with observations made by other investigators (Giot, J.F. et al., (1991) Nucleic Acids Res., 19, 1251 – 8), suggest that Z-plus-R cooperative activation is dependent upon 1) direct binding by R and Z to responsive promoter elements and 2) contributions by cell-specific factors.     

Relating repair susceptibility of carcinogen-damaged DNA with structural distortion and thermodynamic stability

A key issue in the nucleotide excision repair (NER) of bulky carcinogen–DNA adducts is the ability of the NER machinery to recognize and repair certain adducts while failing to repair others. Unrepaired adducts can survive to cause mutations that initiate the carcinogenic process. Benzo[c]phenanthrene (B[c]Ph), a representative fjord region polycyclic aromatic hydrocarbon, can be metabolically activated to the enantiomeric benzo[c]phenanthrene diol epoxides (B[c]PhDEs), (+)-(1S,2R,3R,4S)-3,4- dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]phe nanthrene and the corresponding (–)-(1R,2S,3S,4R) isomer. These react predominantly with adenine residues in DNA to produce the stereoisomeric 1R (+)- and 1S (–)-trans-anti-B[c]Ph-N⁶-dA adducts. Duplexes containing the 1R (+) or 1S (–) B[c]Ph-dA adduct in codon 61 of the human N-ras mutational hotspot sequence CA*A, with B[c]Ph modification at A*, are not repaired by the human NER system. However, the analogous stereoisomeric DNA adducts of the bay region benzo[a]pyrene diol epoxide (B[a]PDE), 10S (+)- and 10R (–)-trans-anti-B[a]P-N⁶-dA, are repaired in the same base sequence. In order to elucidate structural and thermodynamic origins of this phenomenon, we have carried out a 2 ns molecular dynamics simulation for the 1R (+)- and 1S (–)-trans-anti-B[c]Ph-N⁶-dA adducts in an 11mer duplex containing the human N-ras codon 61 sequence, and compared these results with our previous study of the B[a]P-dA adducts in the same sequence. The molecular mechanics Poisson– Boltzmann surface area (MM-PBSA) method was applied to calculate the free energies of the pair of stereoisomeric B[c]Ph-dA adducts, and a detailed structural analysis was carried out. The different repair susceptibilities of the B[a]P-dA adducts and the B[c]Ph-dA adducts can be attributed to different degrees of distortion, stemming from combined effects of differences in the quality of Watson–Crick hydrogen bonding, unwinding, stretching and helix backbone perturbations. These differences are due to the different intrinsic topologies of the rigid, planar bay region adducts versus the twisted, sterically hindered fjord region adducts.     

Two mutations in the tetracycline repressor change the inducer anhydrotetracycline to a corepressor

We report for the first time the in vitro characterization of a reverse tetracycline repressor (revTetR). The dimeric wild-type repressor (TetR) binds to tet operator tetO in the absence of the inducer anhydrotetracycline (atc) to confer tight repression. We have isolated the revTetR G96E L205S mutant, which, contrary to TetR, binds tetO only in the presence of atc. This reverse acting mutant was overproduced and purified. Effector and DNA binding properties were analyzed by EMSA and quantified by fluorescence titration and surface plasmon resonance. The association constant K_A of revTetR for binding of [atcMg]⁺ is ~10⁸ M^–1, four orders of magnitude lower than that of TetR. The affinity of TetR for tetO is 5.6 ± 2 × 10⁹ M^–1 and that for revTetR in the presence of atc is 1 ± 0.2 × 10⁸ M^–1. Both induced forms, the atc-bound TetR and the free revTetR, have the same low affinity of 4 ± 1 × 10⁵ M^–1 for DNA. Therefore, atc does not act as a dimerization agent for revTetR. We discuss the structural differences between TetR and revTetR potentially underlying this reversal of activity.     

A crystallographic study of the binding of 13 metal ions to two related RNA duplexes

Metal ions, and magnesium in particular, are known to be involved in RNA folding by stabilizing secondary and tertiary structures, and, as cofactors, in RNA enzymatic activity. We have conducted a systematic crystallographic analysis of cation binding to the duplex form of the HIV-1 RNA dimerization initiation site for the subtype-A and -B natural sequences. Eleven ions (K⁺, Pb²⁺, Mn²⁺, Ba²⁺, Ca²⁺, Cd²⁺, Sr²⁺, Zn²⁺, Co²⁺, Au³⁺ and Pt⁴⁺) and two hexammines [Co (NH₃)₆]³⁺ and [Ru (NH₃)₆]³⁺ were found to bind to the DIS duplex structure. Although the two sequences are very similar, strong differences were found in their cation binding properties. Divalent cations bind almost exclusively, as Mg²⁺, at ‘Hoogsteen’ sites of guanine residues, with a cation-dependent affinity for each site. Notably, a given cation can have very different affinities for a priori equivalent sites within the same molecule. Surprisingly, none of the two hexammines used were able to efficiently replace hexahydrated magnesium. Instead, [Co (NH₃)₄]³⁺ was seen bound by inner-sphere coordination to the RNA. This raises some questions about the practical use of [Co (NH₃)₆]³⁺ as a [Mg (H₂O)₆]²⁺ mimetic. Also very unexpected was the binding of the small Au³⁺ cation exactly between the Watson–Crick sites of a G-C base pair after an obligatory deprotonation of N1 of the guanine base. This extensive study of metal ion binding using X-ray crystallography significantly enriches our knowledge on the binding of middleweight or heavy metal ions to RNA, particularly compared with magnesium.     

Mg2+-induced triplex formation of an equimolar mixture of poly(rA) and poly(rU)

Magnesium ions strongly influence the structure and biochemical activity of RNA. The interaction of Mg²⁺ with an equimolar mixture of poly(rA) and poly(rU) has been investigated by UV spectroscopy, isothermal titration calorimetry, ultrasound velocimetry and densimetry. Measurements in dilute aqueous solutions at 20°C revealed two differ ent processes: (i) Mg²⁺ binding to unfolded poly(rA)·poly(rU) up to [Mg²⁺]/[phosphate] = 0.25; and (ii) poly(rA)·2poly(rU) triplex formation at [Mg²⁺]/[phosphate] between 0.25 and 0.5. The enthalpies of these two different processes are favorable and similar to each other, ~–1.6 kcal mol^–1 of base pairs. Volume and compressibility effects of the first process are positive, 8 cm³ mol^–1 and 24 × 10^–4 cm³ mol^–1 bar^–1, respectively, and correspond to the release of water molecules from the hydration shells of Mg²⁺ and the polynucleotides. The triplex formation is also accompanied by a positive change in compressibility, 14 × 10^–4 cm³ mol^–1 bar^–1, but only a small change in volume, 1 cm³ mol^–1. A phase diagram has been constructed from the melting experiments of poly(rA)·poly(rU) at a constant K⁺ concentration, 140 mM, and various amounts of Mg²⁺. Three discrete regions were observed, corresponding to single-, double- and triple-stranded complexes. The phase boundary corresponding to the transition between double and triple helical conformations lies near physiological salt concentrations and temperature.     

Complex Adaptations Can Drive the Evolution of the Capacitor [PSI+], Even with Realistic Rates of Yeast Sex

The [PSI⁺] prion may enhance evolvability by revealing previously cryptic genetic variation, but it is unclear whether such evolvability properties could be favored by natural selection. Sex inhibits the evolution of other putative evolvability mechanisms, such as mutator alleles. This paper explores whether sex also prevents natural selection from favoring modifier alleles that facilitate [PSI⁺] formation. Sex may permit the spread of “cheater” alleles that acquire the benefits of [PSI⁺] through mating without incurring the cost of producing [PSI⁺] at times when it is not adaptive. Using recent quantitative estimates of the frequency of sex in Saccharomyces paradoxus, we calculate that natural selection for evolvability can drive the evolution of the [PSI⁺] system, so long as yeast populations occasionally require complex adaptations involving synergistic epistasis between two loci. If adaptations are always simple and require substitution at only a single locus, then the [PSI⁺] system is not favored by natural selection. Obligate sex might inhibit the evolution of [PSI⁺]-like systems in other species.     

An as-1-like motif controls the level of expression of the gene for the pathogenesis-related protein 1a from tobacco

Pathogenesis-related proteins of group 1 (PR-1) are strongly induced in plants by pathogen attack, exposure of the plants to (acetyl)salicylic acid (ASA, SA), and by developmental cues. Functional analysis of the PR-1a promoter identified a region of 139 bp (from -691 to -553) mediating expression of the GUS reporter gene in response to ASA. Inspection of this region revealed two TGACG elements reminiscent of activation sequence-1 (as-1). Recently, as-1 has been reported to be responsive to SA in the context of the CaMV 35S RNA promoter. To address the question of whether the as-1-like sequence may be of functional significance for the expression of the PR-1a gene, gel shift assays were performed with TGA1a, a protein been shown to interact with as-1 in vitro. TGA1a was found to bind to the PR-1a as-1-like sequence with similar specificity and affinity as to as-1. Furthermore, mutations were introduced in the as-1-like sequence in the context of the inducible 906 bp PR-1a promoter which are impaired in binding TGA1a in vitro. Significantly reduced levels of GUS reporter gene activity were obtained with the mutant promoter regions as compared to the wild-type PR-1a promoter in response to all stimuli in transgenic tobacco plants. Yet, mutation of the as-1-like sequence did not abolish induction of reporter gene expression. Taken together, these results suggest that the level of expression of the tobacco PR-1a gene is controlled by an as-1-like sequence motif in the PR-1a upstream region, possibly interacting with a factor related to TGA1a.     

Mutational analyses of dinucleotide and tetranucleotide microsatellites in Escherichia coli: influence of sequence on expansion mutagenesis

Mutagenesis at [GT/CA]₁₀, [TC/AG]₁₁ and [TTCC/AAGG]₉ microsatellite sequences inserted in the herpes simplex virus thymidine kinase (HSV-tk) gene was analyzed in isogenic mutL⁺ and mutL^– Escherichia coli. In both strains, significantly more expansion than deletion mutations were observed at the [TTCC/AAGG]₉ motif relative to either dinucleo­tide motif. As the HSV-tk coding sequence contains an endogenous [G/C]₇ mononucleotide repeat and ~1000 bp of unique sequence, we were able to compare mutagenesis among various sequence motifs. We observed that the relative risk of mutation in E.coli is: [TTCC/AAGG]₉ > [GT/CA]₁₀ ~ [TC/AG]₁₁ > unique ~ [G/C]₇. The mutation frequency varied 1400-fold in mutL⁺ cells between the tetranucleotide motif and the mononucleotide motif, but only 50-fold in mutL^– cells. The [G/C]₇ sequence was destabilized the greatest and the tetranucleotide motif the least by loss of mismatch repair. These results demonstrate that the quantitative risk of mutation at various microsatellites greatly depends on the DNA sequence composition. We suggest alternative models for the production of expansion mutations during lagging strand replication of the [TTCC/AAGG]₉ microsatellite.