Intracellular chloroplast photorelocation in the moss Physcomitrella patens is mediated by phytochrome as well as by a blue-light receptor

 The light-induced intracellular relocation of chloroplasts was examined in red-light-grown protonemal cells of the moss Physcomitrella patens. When irradiated with polarized red or blue light, chloroplast distribution in the cell depended upon the direction of the electrical vector (E-vector) in both light qualities. When the E-vector was parallel to the cross-wall (i.e. perpendicular to the protonemal axis), chloroplasts accumulated along the cross-wall; however, no accumulation along the cross-wall was observed when the E-vector was perpendicular to it (i.e. parallel to the protonemal axis). When a part of the cell was irradiated with a microbeam of red or blue light, chloroplasts accumulated at or avoided the illumination point depending on the fluence rate used. Red light of 0.1–18 W m⁻² and blue light of 0.01–85.5 W m⁻² induced an accumulation response (low-fluence-rate response; LFR), while an avoidance response (high-fluence-rate response; HFR) was induced by red light of 60 W m⁻² or higher and by blue light of 285 W m⁻². The red-light-induced LFR and HFR were nullified by a simultaneous background irradiation of far-red light, whereas the blue-light-induced LFR and HFR were not affected at all by this treatment. These results show, for the first time, that dichroic phytochrome, as well as the dichroic blue-light receptor, is involved in the chloroplast relocation movement in these bryophyte cells. Further, the phytochrome-mediated responses but not the blue-light responses were revealed to be lost when red-light-grown cells were cultured under white light for 2 d. Received: 7 September 1999 / Accepted: 15 October 1999     

Photoinduction of formation of circular structures by microfilaments on chloroplasts during intracellular orientation in protonemal cells of the fernAdiantum capillus-veneris

Summary Changes in the organization of cortical actin microfilaments during phytochrome-mediated and blue light-induced photoorientation of chloroplasts were investigated by rhodamine-phalloidin staining in protonemal cells of the fernAdiantum capillusveneris. Low- and high-fluence rate responses were induced by partial irradiation of individual cells with a microbeam of 20 m in width. In the low-fluence rate responses to red and blue light, a circular structure composed of microfilaments was induced on the chloroplast concentrated in the irradiated region, on the side facing the plasma membrane, as already reported in the case of the low-fluence rate response induced by polarized red or blue light. Such a structure was not observed on the chloroplasts located far from the microbeam. Time-course studies revealed that the structure was induced after the chloroplasts gathered in the illuminated region and that the structure disappeared before chloroplasts moved out of this region when the microbeam was turned off. In the high-fluence rate response to blue light, chloroplasts avoided the irradiated site but accumulated in the shaded area adjacent the edges of microbeam. The circular structure made of microfilaments was also observed on the chloroplasts gathered in the area and it showed the same behavior with respect to its appearance and disappearance during a light/dark regime as in the case of the low-fluence rate response. However, no such circular structure was observed in the high-fluence rate response to red light, in which case the chloroplasts also avoided the illuminated region but no accumulation in the adjacent areas was induced. These results indicate that the circular structure composed of microfilaments may play a role in the anchorage of the chloroplast during intracellular photo-orientation.     

Phytochrome- and blue-light-absorbing pigment-mediated directional movement of chloroplasts in dark-adapted prothallial cells of fernAdiantum as analyzed by microbeam irradiation

When prothalli ofAdiantum capillus-veneris L. were kept for 2 d in the dark, chloroplasts gathered along the anticlinal walls (Kagawa and Wada, 1994, J Plant Res 107: 389–398). In these dark-adapted prothallial cells, irradiation with a microbeam (10 gm in diameter) of red (R) or blue light (B) for 60 s moved the chloroplasts towards the irradiated locus during a subsequent dark period. Chloroplasts located less than 20 gm from the center of the R microbeam (18 J·m^–2) moved towards the irradiated locus. The higher the fluence of the light, the greater the distance from which chloroplasts could be attracted. The B microbeam was less effective than the R microbeam. Chloroplasts started to move anytime up to 20 min after the R stimulus, but with the B microbeam the effect of the stimulus was usually apparent within 10 min after irradiation. The velocity of chloroplast migration was independent of light-fluence in both R and B and was about - 0.3 m·min^–1 between 15 min and 30 min after irradiation. Whole-cell irradiation with far-red light immediately after R- and B-microbeam irradiations demonstrated that these responses were mediated by phytochrome and a blue-light-absorbing pigment, respectively. Sequential treatment with R and B microbeams, whose fluence rates were less than the threshold values when applied separately, resulted in an additive effect and induced chloroplast movement, strongly suggesting that signals from phytochrome and the blue-light-absorbing pigment could interact at some point before the induction of chloroplast movement.Abbreviations B blue light - FR far-red light - IR infrared light - R red light     

Chemical synthesis and translesion replication of a cis-syn cyclobutane thymine-uracil dimer

The cytosine base in DNA undergoes hydrolytic deamination at a considerable rate when UV radiation induces formation of a cyclobutane pyrimidine dimer (CPD) with an adjacent pyrimidine base. We have synthesized a phosphoramidite building block of a cis–syn cyclobutane thymine–uracil dimer (T[]U), which is the deaminated form of the CPD at a TC site, and incorporated it into oligodeoxyribonucleotides. The previously reported method for synthesis of the thymine dimer (T[]T) was applied, using partially protected thymidylyl-(3′–5′)-2′-deoxyuridine as the starting material, and after triplet- sensitized irradiation, the configuration of the base moiety in the major product was determined by NMR spectroscopy. Presence of the cis–syn cyclobutane dimer in the obtained oligonucleotides was confirmed by UV photoreversal and reaction with T4 endonuclease V. Using a 30mer containing T[]U, translesion synthesis by human DNA polymerase η was analyzed. There was no difference in the results between the templates containing T[]T and T[]U and pol η bypassed both lesions with the same efficiency, incorporating two adenylates. This enzyme showed fidelity to base pair formation, but this replication causes a C→T transition because the original sequence is TC.     

In vitro and in vivo modulations of benzo[c]phenanthrene-DNA adducts by DNA mismatch repair system

Benzo[c]phenanthrene dihydrodiol epoxide (B[c] PhDE) is well known as an important environmental chemical carcinogen that preferentially modifies DNA in adenine residues. However, the molecular mechanism by which B[c]PhDE induces tumorigenesis is not fully understood. In this report, we demonstrate that DNA mismatch repair (MMR), a genome maintenance system, plays an important role in B[c]PhDE-induced carcinogensis by promoting apoptosis in cells treated with B[c]PhDE. We show that purified human MMR recognition proteins, MutSα and MutSβ, specifically recognized B[c]PhDE-DNA adducts. Cell lines proficient in MMR exhibited several-fold more sensitivity to killing than cell lines defective in either MutSα or MutLα by B[c]PhDE; the nature of this sensitivity was shown to be due to increased apoptosis. Additionally, wild-type mice exposed to B[c]PhDE had intestinal crypt cells that underwent apoptosis significantly more often than intestinal crypt cells found in B[c]PhDE-treated Msh2^–/– or Mlh1^–/– mice. These findings, combined with previous studies, suggest that the MMR system may serve as a general sensor for chemical-caused DNA damage to prevent damaged cells from mutagenesis and carcinogenesis by promoting apoptosis.     

Extrahelical cytosine bases in DNA duplexes containing d[GCC]n·d[GCC]n repeats: detection by a mechlorethamine crosslinking reaction

The cytosine–cytosine (C–C) pair is one of the least stable DNA mismatch pairs. The bases of the C–C mismatch are only weakly hydrogen bonded, and previous work has shown that, in certain sequence contexts, they can become unstacked from the core helix, and adopt an ‘extrahelical’ location. Here, using DNA duplexes with d[GCC]_n·d[GCC]_n fragments containing C–C mismatches in a 1,4 bp relationship, we show that cytosine bases of different formal mismatch pairs can be crosslinked by mechlorethamine. For example, in the duplex d[CTCTCGCCGCCGCCGTATC]·d[GATACGCCGCCGCCGAGAG], where underlined cytosine bases are present as the formal C–C mismatch pairs C₇–C₃₂, C₁₀–C₂₉ and C₁₃–C₂₆, we show that two mechlorethamine crosslinks form between C₁₃ and C₂₉ and between C₁₀ and C₃₂, in addition to crosslinks at C₇–C₃₂, C₁₀–C₂₉ and C₁₃–C₂₆ (we have reported previously the crosslinking of formal C–C pairs by mechlorethamine). We interpret the formation of the C₁₃–C₂₉ and C₁₀–C₃₂ crosslinks as evidence of an extrahelical location of the crosslinkable cytosines. Such extrahelical cytosine bases have been observed previously for a single C–C mismatch pair (in the so-called E-motif conformation). In the E-motif, the extrahelical cytosines are folded back towards the 5′-end of the duplex, consistent with our crosslinking data, and also consistent with the absence of C₇–C₂₉ and C₁₀–C₂₆ crosslinks in the current work. Hence, our data provide evidence for an extended E-motif DNA (eE-DNA) conformation in short d[GCC]_n·d[GCC]_n repeat fragments, and raise the possibility that such structures might occur in much longer d[GCC]_n·d[GCC]_n repeat tracts.     

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.     

Syntheses and structural studies of calix[4]arene-nucleoside and calix[4]arene-oligonucleotide hybrids

We have synthesized three types of calix[4]arene– nucleoside hybrid efficiently by amide bond formation between the amine functional groups of 1,3-diaminocalix[4]arene and the carboxyl groups of thymidine nucleoside derivatives. X-ray crystallography of a homocoupled calix[4]arene–nucleoside hybrid revealed an interesting hydrogen bonding pattern between thymine bases and the amide linkages. We designed the calix[4]arene–oligonucleotide hybrids (5′-AAAAGATATCAAXTTGATATCTTTT-3′, 5′-T₁₂-X-T₁₂-3′, and 5′-A₁₂-X-T₁₂-3′) to be V-shaped oligodeoxyribonucleotides and synthesized them by using a calix[4]arene–nucleoside hybrid (X) as a key building block. Thermal denaturation experiments, monitored by UV spectroscopy at 260 and 284 nm, and circular dichroism spectra of the calix[4]arene–oligonucleotide hybrids suggest that the modified oligonucleotides indeed adopt V-shaped conformations, making them suitable for use as building blocks in the construction of programmed oligonucleotide nanostructures.     

Ensemble and single-molecule fluorescence spectroscopic study of the binding modes of the bis-benzimidazole derivative Hoechst 33258 with DNA

Ensemble and single-molecule fluorescence measurements of 2′-(4-hydroxyphenyl)-5-[5-(4-methylpiperazine-1-yl) benzimidazo-2-yl]-benzimidazole (H-258)– calf thymus (CT) DNA complexes at various [H-258]/[DNA bp] ratios were performed to elucidate the binding of H-258 with DNA. Upon binding to double-stranded CT DNA (CT ds DNA) at a [H-258]/[DNA bp] ratio of 0.05 the relative fluorescence quantum yield, Φ_f, of H-258 increases from 0.02 to 0.58. The fluorescence decay can be fitted almost by a mono-exponential model with a lifetime of ~3.6 ns. This indicates that H-258 binds almost quantitatively in the minor groove of DNA at low [H-258]/[DNA bp] ratios. With increasing [H-258]/[DNA bp] ratios, e.g. 0.15 and 0.20, the fluorescence quantum yield of H-258 decreases to 0.28 and 0.19, respectively. Fitting of the fluorescence decays measured for higher [H-258]/[DNA bp] ratios reveals the presence of additional shorter fluorescence lifetime components in the range of 0.5–2.0 ns. Our results suggest that H-258 partially intercalates in G:C sequences at higher [H-258]/[DNA bp] ratios reflected by a lifetime component of 1.5–2 ns. In addition, stacking or adsorption of H-258 molecules on DNA occurs at higher [H-258]/[DNA bp] ratios. These molecules exhibit a short fluorescence lifetime of ~500 ps and are more exposed to the aqueous environment. Fluorescence transients of the intensity and lifetime of single H-258 CT ds DNA demonstrate that weakly (unspecific) bound H-258 molecules exhibit a shorter fluorescence lifetime and a strongly reduced photostability.     

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.     

Blue light-induced chloroplast relocation in Arabidopsis thaliana as analyzed by microbeam irradiation

Chloroplast relocation in mesophyll cells of Arabidopsis thaliana was observed microscopically and analyzed by microbeam irradiation. Chloroplasts located along the anticlinal walls in dark-adapted cells. When part of a cell was irradiated with a microbeam of high fluence rate blue light (B) simultaneously with background red light (R) on the whole cell, the chloroplasts moved towards the B-irradiated area, but did not enter the beam. The background R illumination activated cytoplasmic motility as well as chloroplast movement. Without R illumination, there was little chloroplast relocation. In light-adapted cells in which the chloroplasts were spread over the cell surface perpendicular to the incident light, R-illumination had the same effect. Under background R, the chloroplasts moved out of the area irradiated with a B microbeam of 8 or 30 W m(-2) (avoidance response), but chloroplasts outside the beam moved towards the area irradiated with the B microbeam (accumulation response). These results suggest that the signals for accumulation and avoidance responses were generated in a single cell by high fluence rate B. cry1cry2, npq1 and nph1 mutants showed B-induced chloroplast relocation. Both the accumulation and avoidance responses were observed in all the mutants, although in the nph1 mutant, the sensitivity of accumulation movement was slightly lower than that of the wild type. We discuss the possible photoreceptor for B-induced chloroplast relocation.     

Dynamics of the B-A transition of DNA double helices

Although the transition from the B-DNA double helix to the A-form is essential for biological function, as shown by the existence of the A-form in many protein–DNA complexes, the dynamics of this transition has not been resolved yet. According to molecular dynamics simulations the transition is expected in the time range of a few nanoseconds. The B–A transition induced by mixing of DNA samples with ethanol in stopped flow experiments is complete within the deadtime, showing that the reaction is faster than ~0.2 ms. The reaction was resolved by an electric field jump technique with induction of the transition by a dipole stretching force driving the A- to the B-form. Poly[d(A-T)] was established as a favourable model system, because of a particularly high cooperativity of the transition and because of a spectral signature allowing separation of potential side reactions. The time constants observed in the case of poly[d(A-T)] with ~1600 bp are in the range around 10 µs. An additional process with time constants of ~100 µs is probably due to nucleation. The same time constants (within experimental accuracy ±10%) were observed for a poly[d(A-T)] sample with ~70 bp. Under low salt conditions commonly used for studies of the B–A transition, the time constants are almost independent of the ionic strength. The experimental data show that a significant activation barrier exists in the B–A transition and that the helical states are clearly separated from each other, in contrast to predictions by molecular dynamics simulations.     

The Lantibiotic Nisin Induces Transmembrane Movement of a Fluorescent Phospholipid

Nisin is a pore-forming antimicrobial peptide. The capacity of nisin to induce transmembrane movement of a fluorescent phospholipid in lipid vesicles was investigated. Unilamellar phospholipid vesicles that contained a fluorescent phospholipid (1-acyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]caproyl}-sn-glycero-3-phosphocholine) in the inner leaflet of the bilayer were used. Nisin-induced movement of the fluorescent phospholipid from the inner leaflet to the outer leaflet of the membrane reached stable levels, which were dependent on the concentration of nisin added. The rate constant k of this nisin-induced transmembrane movement increased with the nisin concentration but was not dependent on temperature within the range of 5 to 30°C. In contrast, the rate constant of movement of fluorescent phospholipid from vesicle to vesicle strongly depended on temperature. The data indicate that nisin transiently disturbs the phospholipid organization of the target membrane.     

The solution structure of [d(CGC)r(aaa)d(TTTGCG)](2): hybrid junctions flanked by DNA duplexes

The solution structure and hydration of the chimeric duplex [d(CGC)r(aaa)d(TTTGCG)]₂, in which the central hybrid segment is flanked by DNA duplexes at both ends, was determined using two-dimensional NMR, simulated annealing and restrained molecular dynamics. The solution structure of this chimeric duplex differs from the previously determined X-ray structure of the analogous B-DNA duplex [d(CGCAAATTTGCG)]₂ as well as NMR structure of the analogous A-RNA duplex [r(cgcaaauuugcg)]₂. Long-lived water molecules with correlation time τ_c longer than 0.3 ns were found close to the RNA adenine H2 and H1′ protons in the hybrid segment. A possible long-lived water molecule was also detected close to the methyl group of 7T in the RNA–DNA junction but not with the other two thymines (8T and 9T). This result correlates with the structural studies that only DNA residue 7T in the RNA–DNA junction adopts an O4′-endo sugar conformation, while the other DNA residues including 3C in the DNA–RNA junction, adopt C1′-exo or C2′-endo conformations. The exchange rates for RNA C2′-OH were found to be ~5–20 s^–1. This slow exchange rate may be due to the narrow minor groove width of [d(CGC)r(aaa)d(TTTGCG)]₂, which may trap the water molecules and restrict the dynamic motion of hydroxyl protons. The minor groove width of [d(CGC)r(aaa)d(TTTGCG)]₂ is wider than its B-DNA analog but narrower than that of the A-RNA analog. It was further confirmed by its titration with the minor groove binding drug distamycin. A possible 2:1 binding mode was found by the titration experiments, suggesting that this chimeric duplex contains a wider minor groove than its B-DNA analog but still narrow enough to hold two distamycin molecules. These distinct structural features and hydration patterns of this chimeric duplex provide a molecular basis for further understanding the structure and recognition of DNA·RNA hybrid and chimeric duplexes.     

Error-prone lesion bypass by human DNA polymerase eta

DNA lesion bypass is an important cellular response to genomic damage during replication. Human DNA polymerase η (Polη), encoded by the Xeroderma pigmentosum variant (XPV) gene, is known for its activity of error-free translesion synthesis opposite a TT cis-syn cyclobutane dimer. Using purified human Polη, we have examined bypass activities of this polymerase opposite several other DNA lesions. Human Polη efficiently bypassed a template 8-oxoguanine, incorporating an A or a C opposite the lesion with similar efficiencies. Human Polη effectively bypassed a template abasic site, incorporating an A and less frequently a G opposite the lesion. Significant –1 deletion was also observed when the template base 5′ to the abasic site is a T. Human Polη partially bypassed a template (+)-trans-anti-benzo[a]pyrene-N²-dG and predominantly incorporated an A, less frequently a T, and least frequently a G or a C opposite the lesion. This specificity of nucleotide incorporation correlates well with the known mutation spectrum of (+)-trans-anti-benzo[a]pyrene-N²-dG lesion in mammalian cells. These results show that human Polη is capable of error-prone translesion DNA syntheses in vitro and suggest that Polη may bypass certain lesions with a mutagenic consequence in humans.     

Response of human REV1 to different DNA damage: preferential dCMP insertion opposite the lesion

REV1 functions in the DNA polymerase ζ mutagenesis pathway. To help understand the role of REV1 in lesion bypass, we have examined activities of purified human REV1 opposite various template bases and several different DNA lesions. Lacking a 3′→5′ proofreading exonuclease activity, purified human REV1 exhibited a DNA polymerase activity on a repeating template G sequence, but catalyzed nucleotide insertion with 6-fold lower efficiency opposite a template A and 19–27-fold lower efficiency opposite a template T or C. Furthermore, dCMP insertion was greatly preferred regardless of the specific template base. Human REV1 inserted a dCMP efficiently opposite a template 8-oxoguanine, (+)-trans-anti-benzo[a]pyrene-N ²-dG, (–)-trans-anti-benzo[a]pyrene-N ²-dG and 1,N ⁶-ethenoadenine adducts, very inefficiently opposite an acetylaminofluorene-adducted guanine, but was unresponsive to a template TT dimer or TT (6–4) photoproduct. Surprisingly, the REV1 specificity of nucleotide insertion was very similar in response to different DNA lesions with greatly preferred C insertion and least frequent A insertion. By combining the dCMP insertion activity of human REV1 with the extension synthesis activity of human polymerase κ, bypass of the trans-anti-benzo[a]pyrene-N ² -dG adducts and the 1,N ⁶-ethenoadenine lesion was achieved by the two-polymerase two-step mechanism. These results suggest that human REV1 is a specialized DNA polymerase that may contribute to dCMP insertion opposite many types of DNA damage during lesion bypass.     

RNA structure-dependent uncoupling of substrate recognition and cleavage by Escherichia coli ribonuclease III

Members of the ribonuclease III superfamily of double-strand-specific endoribonucleases participate in diverse RNA maturation and decay pathways. Ribonuclease III of the gram-negative bacterium Escherichia coli processes rRNA and mRNA precursors, and its catalytic action can regulate gene expression by controlling mRNA translation and stability. It has been proposed that E.coli RNase III can function in a non-catalytic manner, by binding RNA without cleaving phosphodiesters. However, there has been no direct evidence for this mode of action. We describe here an RNA, derived from the T7 phage R1.1 RNase III substrate, that is resistant to cleavage in vitro by E.coli RNase III but retains comparable binding affinity. R1.1[CL3B] RNA is recognized by RNase III in the same manner as R1.1 RNA, as revealed by the similar inhibitory effects of a specific mutation in both substrates. Structure-probing assays and Mfold analysis indicate that R1.1[CL3B] RNA possesses a bulge– helix–bulge motif in place of the R1.1 asymmetric internal loop. The presence of both bulges is required for uncoupling. The bulge–helix–bulge motif acts as a ‘catalytic’ antideterminant, which is distinct from recognition antideterminants, which inhibit RNase III binding.     

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.     

Modulation of plasma protein binding and in vivo liver cell uptake of phosphorothioate oligodeoxynucleotides by cholesterol conjugation

Several studies have shown improved efficacy of cholesteryl-conjugated phosphorothioate antisense oligodeoxynucleotides. To gain insight into the mechanisms of the improved efficacy in vivo, we investigated the disposition of ISIS-9388, the 3′-cholesterol analog of the ICAM-1-specific phosphoro­thioate oligodeoxynucleotide ISIS-3082, in rats. Intravenously injected [³H]ISIS-9388 was cleared from the circulation with a half-life of 49.9 ± 2.2 min (ISIS-3082, 23.3 ± 3.8 min). At 3 h after injection, the liver contained 63.7 ± 3.3% of the dose. Compared to ISIS-3082, the hepatic uptake of ISIS-9388 is ~2-fold higher. Endothelial, Kupffer and parenchymal cells accounted for 45.7 ± 5.7, 33.0 ± 5.9 and 21.3 ± 2.6% of the liver uptake of [³H]ISIS-9388, respectively, and intracellular concentrations of ~2, 75 and 50 µM, respectively, could be reached in these cells (1 mg/kg dose). Preinjection with polyinosinic acid or poly­adenylic acid reduced the hepatic uptake of [³H]ISIS-9388, which suggests the involvement of (multiple) scavenger receptors. Size exclusion chromatography of mixtures of the oligonucleotides and rat plasma indicated that ISIS-9388 binds to a larger extent to high molecular weight proteins than ISIS-3082. Analysis by agarose gel electrophoresis indicated that ISIS-9388 binds more tightly to plasma proteins than ISIS-3082. The different interaction of the oligonucleotides with plasma proteins possibly explains their different dispositions. We conclude that cholesterol conjugation results in high accumulation of phosphorothioate oligodeoxynucleotides in various liver cell types, which is likely to be beneficial for antisense therapy of liver-associated diseases.     

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.