Physico-chemical model for DNA alkaline elution: New experimental evidence and differential role of DNA length,chain flexibility and superpacking

For a better understanding of data provided by DNA alkaline elution technique, a new analytical model has been developed which takes into consideration both the physicochemical properties of in situ DNA strand (length and flexibility/superpacking) and the geometric and hydrodynamic configuration of the elution apparatus (flow and filter conditions). Simulation by this model of experimental data previously obtained before and after carcinogens administration, has shown that for constant flow and filter conditions elution profiles are dependent, not only from DNA molecular weight, but also from a parameter critically related to modifications in chain flexibility/superpacking. This has been confirmed by several independent observations, including the time-dependent changes in non-denaturing lysing solution monitored by hydroxylapatite and alkaline elution techniques.     

Investigation of 13C relaxation and nuclear Overhauser enhancement parameters for heparinoid systems: comparison with data from dextrans

Data are presented for ¹³C spin-lattice (T₁) relaxation times and nuclear Overhauser enhancement (NOE) values in a range of heparinoid glycosaminoglycans. These paramaters are compared with T₁and NOE values for simple dextrans of $\text{M}{}_{\mathrm{<mtext>W</mtext>}}\text{= 19 900, 40 000}\text{and}\text{150 000}$. For the heparinoid molecules, significant and consistent differences in relaxation times are observed for different ring carbons, for which simple models do not provide an adequate explanation. No such variations are found for the dextrans. Both families of polysaccharides exhibit NOE values significantly reduced from the theoretical maximum. These changes are discussed in terms of molecular rotational correlation times (τ_c) which are similar in magnitude to the resonance frequencies used for n.m.r. measurements. It is concluded that for the n.m.r. investigation of polysaccharides at high field strengths (${}^{13}\text{C resonance frequencies > 25 MHz}$) considerable economy of time will be achieved by the use of INEPT experiments rather than conventional Overhauser signal enhancement via proton-noise decoupling.     

Kinetics of DNA Unwinding by the RecD2 Helicase from Deinococcus radiodurans

RecD2 from Deinococcus radiodurans is a superfamily 1 DNA helicase that is homologous to the Escherichia coli RecD protein but functions outside the context of RecBCD enzyme. We report here on the kinetics of DNA unwinding by RecD2 under single and multiple turnover conditions. There is little unwinding of 20-bp substrates by preformed RecD2-dsDNA complexes when excess ssDNA is present to trap enzyme molecules not bound to the substrate. A shorter 12-bp substrate is unwound rapidly under single turnover conditions. The 12-bp unwinding reaction could be simulated with a mechanism in which the DNA is unwound in two kinetic steps with rate constant of k_unw = 5.5 s⁻¹ and a dissociation step from partially unwound DNA of k_off = 1.9 s⁻¹. These results indicate a kinetic step size of about 3–4 bp, unwinding rate of about 15–20 bp/s, and low processivity (p = 0.74). The reaction time courses with 20-bp substrates, determined under multiple turnover conditions, could be simulated with a four-step mechanism and rate constant values very similar to those for the 12-bp substrate. The results indicate that the faster unwinding of a DNA substrate with a forked end versus only a 5′-terminal single-stranded extension can be accounted for by a difference in the rate of enzyme binding to the DNA substrates. Analysis of reactions done with different RecD2 concentrations indicates that the enzyme forms an inactive dimer or other oligomer at high enzyme concentrations. RecD2 oligomers can be detected by glutaraldehyde cross-linking but not by size exclusion chromatography.     

A circular channel crucible oscillating viscometer: Detection of DNA damage induced in vivo by exceedingly small doses of dimethylnitrosamine

A new oscillating crucible viscometer, having a U-shaped circular channel, is described. The damping coefficient δ is lowered by an increase of the viscosity η. The instrument described here allows the solution to come in contact with inert plastic only. At all steps of its preparation and during viscosity measurements, giant DNA from rat liver nuclei was maintained at shear stresses around 10^?4 dynes cm^?2. Viscosity was studied as a function of surface tension, DNA concentration and shear stress. It was found that under our experimental conditions it was possible to obtain meaningful values for reduced viscosity, η_red, practically identical to intrinsic viscosity [η]. Rat liver nuclei are incubated in an alkaline lysing solution (pH 12.5; 22 °C): they are lysed immediately and the released DNA starts to uncoil. The viscosity of solutions of this giant DNA increases very slowly with time, reaching a maximum only after about ten hours. The process was accelerated by single-stranded breaks arising from methylation of DNA in vivo with dimethylnitrosamine. It was found that the time of DNA disentanglement was sensitive to an exceedingly small number of breaks. We think that we were able to measure molecular weights around the length of the single strand of an average chromosome (M_n 5 × 10¹⁰). An empirical relation between molecular weight and reduced viscosity after complete disentanglement was also established, as a linear log-log plot, covering a molecular weight range between 10⁸ and 2.5 × 10¹⁰. It is suggested that the viscosimetric evaluation of DNA disentanglement is probably the most sensitive method for studying DNA damage induced “in vivo” by chemical carcinogens.     

Sequential resonance assignments in protein 1H nuclear magnetic resonance spectra: Computation of sterically allowed proton-proton distances and statistical analysis of proton-proton distances in single crystal protein conformations

Two different, theoretical studies of intramolecular proton-proton distances in polypeptide chains are described. Firstly, the distances between amide, C^α and C^β protons of neighbouring residues in the amino acid sequence, which correspond to the sterically allowed values for the dihedral angles φ_i, ψ_i and χ_i¹, were computed. Secondly, the frequency with which short distances occur between amide, C^α and C^β protons of neighbouring and distant residues in the amino acid sequence were statistically evaluated in a representative sample of globular protein crystal structures. Both approaches imply that semi-quantitative measurements of short, non-bonding proton-proton distances, e.g. by nuclear Overhauser experiments, should present a reliable and generally applicable method for sequential, individual resonance assignments in protein ¹H nuclear magnetic resonance spectra. Similar calculations imply that corresponding distance measurements can be used for resonance assignments in the side-chains of the aromatic amino acid residues, asparagine and glutamine, where the complete spin systems cannot usually be identified from through-bond spin-spin coupling connectivities.     

Quantitative internuclear distancesvia two-dimensional nuclear magnetic resonance spectra: A test case and a DNA octamer duplex

Two-dimensional proton nuclear magnetic resonance nuclear Overhauser effect experiments have been performed at a series of mixing times on proflavine and on a DNA octamer duplex [d-(GGAATTCC)]₂ in solution. Using the complete matrix approach recently explored theoretically (Keepers and James, 1984), proton-proton internuclear distances were determined quantitatively for proflavine from the two-dimensional nuclear Overhauser effect results. Since proflavine is a rigid molecule with X-ray crystal structure determined, interproton distances obtained from the two-dimensional nuclear Overhauser effect experiments in solution can be compared with those for the crystalline compound agreement is better than 10 %. Experimental two-dimensional nuclear Overhauser effect spectral data for [d-(GGAATTCC)]₂ were analyzed by comparison with theoretical two-dimensional nuclear Overhauser effect spectra at each mixing time calculated using the complete 70 × 70 relaxation matrix. The theoretical spectra were calculated using two structures: a standard B-form DNA structure and an energy-minimized structure based on similarity of the octamer's six internal residues with those of [d-(CGCGAATTCGCG)]₂, for which the crystal structure has been determined. Neither the standard B-DNA nor the energy-minimized structure yield theoretical two-dimensional nuclear Overhauser effect spectra which accurately reproduce all experimental peak intensities. But many aspects of the experimental spectra can be represented by both the B-DNA and the energy-minimized structure. In general, the energy-minimized structure yields theoretical two-dimensional nuclear Overhauser effect spectra which mimic many, if not all, features of the experimental, spectra including structural characteristics at the purine-pyrimidine junction.     

Revisiting absorbance at 230 nm as a protein unfolding probe

Thermodynamic stability and unfolding kinetics of proteins are typically determined by monitoring protein unfolding with spectroscopic probes, such as circular dichroism (CD) and fluorescence. UV absorbance at 230 nm (A₂₃₀) is also known to be sensitive to protein conformation. However, its feasibility for quantitative analysis of protein energetics has not been assessed. Here we evaluate A₂₃₀ as a structural probe to determine thermodynamic stability and unfolding kinetics of proteins. By using Escherichia coli maltose binding protein (MBP) and E. coli ribonuclease H (RNase H) as our model proteins, we monitored their unfolding in urea and guanidinium chloride with A₂₃₀. Significant changes in A₂₃₀ were observed with both proteins on unfolding in the chemical denaturants. The global stabilities were successfully determined by measuring the change in A₂₃₀ in varying concentrations of denaturants. Also, unfolding kinetics was investigated by monitoring the change in A₂₃₀ under denaturing conditions. The results were quite consistent with those determined by CD. Unlike CD, A₂₃₀ allowed us to monitor protein unfolding in a 96-well microtiter plate with a UV plate reader. Our finding suggests that A₂₃₀ is a valid and convenient structural probe to determine thermodynamic stability and unfolding kinetics of proteins with many potential applications.     

A Parallel Quadruplex DNA Is Bound Tightly but Unfolded Slowly by Pif1 Helicase

DNA sequences that can form intramolecular quadruplex structures are found in promoters of proto-oncogenes. Many of these sequences readily fold into parallel quadruplexes. Here we characterize the ability of yeast Pif1 to bind and unfold a parallel quadruplex DNA substrate. We found that Pif1 binds more tightly to the parallel quadruplex DNA than single-stranded DNA or tailed duplexes. However, Pif1 unwinding of duplexes occurs at a much faster rate than unfolding of a parallel intramolecular quadruplex. Pif1 readily unfolds a parallel quadruplex DNA substrate in a multiturnover reaction and also generates some product under single cycle conditions. The rate of ATP hydrolysis by Pif1 is reduced when bound to a parallel quadruplex compared with single-stranded DNA. ATP hydrolysis occurs at a faster rate than quadruplex unfolding, indicating that some ATP hydrolysis events are non-productive during unfolding of intramolecular parallel quadruplex DNA. However, product eventually accumulates at a slow rate.     

Analysis of the distribution of DNA repair patches in the DNA-nuclear matrix complex from human cells

The distribution of ultraviolet-induced repair patches along DNA loops attached to the nuclear matrix, was investigated by digestion with DNA-degrading enzymes and neutral sucrose gradient centrifugation. When DNA was gradually removed by DNAase 1, pulse label incorporated by ultraviolet-irradiated cells during 10 min in the presence of hydroxyurea or hydroxyurea/arabinosylcytosine showed similar degradation kinetics as prelabelled DNA. No preferential association of pulse label with the nuclear matrix was observed, neither within 30 min nor 13 h after irradiation. When the pulse label was incorporated by replicative synthesis under the same conditions, a preferential association of newly-synthesized DNA with the nuclear matrix was observed. Single-strand specific digestion with nuclease S₁ of nuclear lysates from ultraviolet-irradiated cells, pulse labelled in the presence of hydroxyurea/arabinosylcytosine, caused a release of about 70% of the prelabelled DNA and 90% of the pulse-labelled DNA from the rapidly sedimenting material in sucrose gradients. The results suggest no specific involvement of the nuclear matrix in repair synthesis, a random distribution of repair patches along the DNA loops, and simultaneously multiple incision events per DNA loop.     

The quinternary chromatin-DNA structure

Nuclear DNA-space images from Feulgen-stained HeLa cells synchronized at 1, 3, 5, 8, 12, 15, and 18 h following mitosis are digitized and their densitometric-geometric patterns are analyzed by means of a Quantimet 720-D image analyzer on line with a PDP11/40 computer. Frequency distributions of picture point optical densities for the phases and subphases as seen in nuclear images show that DNA packing changes are evident by means of ordinary optical microscopy. Radii of gyration of the images, and optical density profiles and distributions for several squashes of similar cells reveal that in particular instances chromatin DNA is distributed mostly towards the periphery, and usually with high circular isotropy. Cross power spectra of individual scan lines suggest the existence of higher order “quinternary” periodic structure for chromatin that modulates during the cell cycle. Three-dimensional reconstruction of 2- μm sections of intact, Feulgen-stained mammalian tumor tissue show stainable material only toward the nuclear perimeter and not in the center (compatible with the evidence that initial thymidine incorporation in HeLa cells is generally at the nuclear border). Densitometric properties of reconstructed interphase chromatin-DNA bodies are highly coupled with similar properties of the whole nucleus, showing that a more condensed nucleus is always accompanied by a more condensed interphase chromatin DNA. The effect of micrococcal nuclease digestion on the digitized nuclear images is also presented. All the above data are then discussed in terms of a quinternary chromatin-DNA structure and its modulation during the cell cycle.     

Substrate-specific inhibition of RecQ helicase

The RecQ helicases constitute a small but highly conserved helicase family. Proteins in this family are of particular interest because they are critical to maintenance of genomic stability in prokaryotes and eukaryotes. Eukaryotic RecQ helicase family members have been shown to unwind not only DNA duplexes but also DNAs with alternative structures, including structures stabilized by G quartets (G4 DNAs). We report that Escherichia coli RecQ can also unwind G4 DNAs, and that unwinding requires ATP and divalent cation. RecQ helicase is comparably active on duplex and G4 DNA substrates, as measured by direct comparison of protein activity and by competition assays. The porphyrin derivative, N-methyl mesoporphyrin IX (NMM), is a highly specific inhibitor of RecQ unwinding activity on G4 DNA but not duplex DNA: the inhibition constant (K_i) for NMM inhibition of G4 DNA unwinding is 1.7 µM, approximately two orders of magnitude below the K_i for inhibition of duplex DNA unwinding (>100 µM). NMM may therefore prove to be a valuable compound for substrate-specific inhibition of other RecQ family helicases in vitro and in vivo.     

DNA breakage detection-fish (DBD-FISH): effect of unwinding time

DBD-FISH is a new procedure that allows detection and quantification of DNA breakage in situ within specific DNA target sites. Cells embedded in an agarose matrix on a slide are treated in an alkaline unwinding solution to transform DNA breaks into single-stranded DNA (ssDNA). After removal of proteins, DNA probes are hybridized and detected. DNA breaks increase the ssDNA and relax supercoiling of DNA loops, so more probe hybridizes, thereby increasing the surface area and fluorescence intensity of the FISH signal. The probe selects the chromatin area to be analysed.In order to restrict the extension of unwound ssDNA to a region closer to the origin of the DNA break, human leukocytes were processed for DBD-FISH with a whole genome probe, after a 10 Gy dose of X-rays, for various unwinding times: 5, 2 min and 30s. Two cell populations were detected after 30s, but not with the 5 or 2 min unwinding times. One cell group had small to medium haloes corresponding to the relaxation of DNA supercoiling after DAPI staining, and strong DBD-FISH labelling of induced DNA breaks, whereas the other cell group showed big haloes of DNA loop unfolding and an absence of DBD-FISH labelling. The latter group was similar to cells processed by DBD-FISH without the unwinding step. Thus, they should correspond to cells unaffected by the alkaline unwinding solution, possibly because very brief unwinding times do not allow the diffusion of the alkali into the cells deep within the gel, thus biasing the results. Taking this into account, 2 min seems to be the minimum unwinding time required for an accurate detection of a signal by DBD-FISH.     

Liver Nuclear Nadph-Cytochrome P-450 Reductase May be Involved in Redox Cycling of Bleomycin-Fe(III), Oxy Radical Formation and DNA Damage

When NADPH-cytochrome P-450 reductase isolated from rat liver microsomes was aerobically incubated with bleomycin, FeCl₃, NADPH and DNA parallel NADPH and oxygen were consumed and malondialdehyde was formed. A similar parallelism of NADPH- and oxygen-consumption and malondialdehyde formation was observed when ceil nuclei isolated from rat liver were incubated under the same conditions. The formation of malondialdehyde which was identified by HPLC and which was most likely released from oxidative cleavage of deoxyribose of nuclear DNA required oxygen, bleomycin, FeCl₃ and NADPH. This indicates that a nuclear NADPH-enzyme, presumably NADPH-cytochrome P-450 reductase, is able to redox cycle a bleomycin-iron-complex which in the reduced form can activate oxygen to a DNA-damaging reactive species. The data suggest that the activity of this enzyme in the cell nucleus could play an important role in the cytotoxicity of bleomycin in tumor cells.     

G-quadruplex and G-rich sequence stimulate Pif1p-catalyzed downstream duplex DNA unwinding through reducing waiting time at ss/dsDNA junction

Alternative DNA structures that deviate from B-form double-stranded DNA such as G-quadruplex (G4) DNA can be formed by G-rich sequences that are widely distributed throughout the human genome. We have previously shown that Pif1p not only unfolds G4, but also unwinds the downstream duplex DNA in a G4-stimulated manner. In the present study, we further characterized the G4-stimulated duplex DNA unwinding phenomenon by means of single-molecule fluorescence resonance energy transfer. It was found that Pif1p did not unwind the partial duplex DNA immediately after unfolding the upstream G4 structure, but rather, it would dwell at the ss/dsDNA junction with a ‘waiting time’. Further studies revealed that the waiting time was in fact related to a protein dimerization process that was sensitive to ssDNA sequence and would become rapid if the sequence is G-rich. Furthermore, we identified that the G-rich sequence, as the G4 structure, equally stimulates duplex DNA unwinding. The present work sheds new light on the molecular mechanism by which G4-unwinding helicase Pif1p resolves physiological G4/duplex DNA structures in cells.     

DNA base excision repair activities and pathway function in mitochondrial and cellular lysates from cells lacking mitochondrial DNA

Mitochondrial DNA (mtDNA) contains higher steady-state levels of oxidative damage and mutates at rates significantly greater than nuclear DNA. Oxidative lesions in mtDNA are removed by a base excision repair (BER) pathway. All mtDNA repair proteins are nuclear encoded and imported. Most mtDNA repair proteins so far discovered are either identical to nuclear DNA repair proteins or isoforms of nuclear proteins arising from differential splicing. Regulation of mitochondrial BER is therefore not expected to be independent of nuclear BER, though the extent to which mitochondrial BER is regulated with respect to mtDNA amount or damage is largely unknown. Here we have measured DNA BER activities in lysates of mitochondria isolated from human 143B TK^– osteosarcoma cells that had been depleted of mtDNA (ρ⁰) or not (wt). Despite the total absence of mtDNA in the ρ⁰ cells, a complete mitochondrial BER pathway was present, as demonstrated using an in vitro assay with synthetic oligonucleotides. Measurement of individual BER protein activities in mitochondrial lysates indicated that some BER activities are insensitive to the lack of mtDNA. Uracil and 8-oxoguanine DNA glycosylase activities were relatively insensitive to the absence of mtDNA, only about 25% reduced in ρ⁰ relative to wt cells. Apurinic/apyrimidinic (AP) endonuclease and polymerase γ activities were more affected, 65 and 45% lower, respectively, in ρ⁰ mitochondria. Overall BER activity in lysates was also about 65% reduced in ρ⁰ mitochondria. To identify the limiting deficiencies in BER of ρ⁰ mitochondria we supplemented the BER assay of mitochondrial lysates with pure uracil DNA glycosylase, AP endonuclease and/or the catalytic subunit of polymerase γ. BER activity was stimulated by addition of uracil DNA glycosylase and polymerase γ. However, no addition or combination of additions stimulated BER activity to wt levels. This suggests that an unknown activity, factor or interaction important in BER is deficient in ρ⁰ mitochondria. While nuclear BER protein levels and activities were generally not altered in ρ⁰ cells, AP endonuclease activity was substantially reduced in nuclear and in whole cell extracts. This appeared to be due to reduced endogenous reactive oxygen species (ROS) production in ρ⁰ cells, and not a general dysfunction of ρ⁰ cells, as exposure of cells to ROS rapidly stimulated increases in AP endonuclease activities and APE1 protein levels.     

Detection of DNA damage induced in vivo by a cross-linking agent with a circular channel crucible oscillating viscometer

DNA damage induced in vivo by the cross-linking agent mitomycin C (MMC) was investigated with a new oscillating crucible viscometer. Viscosity was measured by lysing rat liver nuclei in an alkaline lysing solution (pH 12.5; 25 degrees C). In control samples the viscosity increased very slowly with time, reaching a plateau only after 10-12 h. The process was accelerated and the maximum viscosity was decreased by alkaline single-stranded breaks arising from methylation and subsequent depurination of DNA in vitro with dimethylsulphate (DMS). MMC, when given alone, had no evident effect on the time needed for reaching plateau viscosity but it induced a small increase in maximum viscosity. When MMC was given in association with DMS, the time of disentanglement remained unchanged (accelerated) but maximum viscosity was increased in a dose dependent way. We conclude that these data clearly confirm that the slow steady increase of the viscosity of control DNA with time reflects mainly the process of unwinding of the two strands. The speed of this process seems to depend only from the number of unwinding points in DNA (breaks).     

Influence of DNA end structure on the mechanism of initiation of DNA unwinding by the Escherichia coli RecBCD and RecBC helicases

Escherichiacoli RecBCD is a bipolar DNA helicase possessing two motor subunits (RecB, a 3′-to-5′ translocase, and RecD, a 5′-to-3′ translocase) that is involved in the major pathway of recombinational repair. Previous studies indicated that the minimal kinetic mechanism needed to describe the ATP-dependent unwinding of blunt-ended DNA by RecBCD in vitro is a sequential n-step mechanism with two to three additional kinetic steps prior to initiating DNA unwinding. Since RecBCD can “melt out” ∼ 6 bp upon binding to the end of a blunt-ended DNA duplex in a Mg²⁺-dependent but ATP-independent reaction, we investigated the effects of noncomplementary single-stranded (ss) DNA tails [3′-(dT)₆ and 5′-(dT)₆ or 5′-(dT)₁₀] on the mechanism of RecBCD and RecBC unwinding of duplex DNA using rapid kinetic methods. As with blunt-ended DNA, RecBCD unwinding of DNA possessing 3′-(dT)₆ and 5′-(dT)₆ noncomplementary ssDNA tails is well described by a sequential n-step mechanism with the same unwinding rate (mk_U = 774 ± 16 bp s^− 1) and kinetic step size (m = 3.3 ± 1.3 bp), yet two to three additional kinetic steps are still required prior to initiation of DNA unwinding (k_C = 45 ± 2 s^− 1). However, when the noncomplementary 5′ ssDNA tail is extended to 10 nt [5′-(dT)₁₀ and 3′-(dT)₆], the DNA end structure for which RecBCD displays optimal binding affinity, the additional kinetic steps are no longer needed, although a slightly slower unwinding rate (mk_U = 538 ± 24 bp s^− 1) is observed with a similar kinetic step size (m = 3.9 ± 0.5 bp). The RecBC DNA helicase (without the RecD subunit) does not initiate unwinding efficiently from a blunt DNA end. However, RecBC does initiate well from a DNA end possessing noncomplementary twin 5′-(dT)₆ and 3′-(dT)₆ tails, and unwinding can be described by a simple uniform n-step sequential scheme, without the need for the additional k_C initiation steps, with a similar kinetic step size (m = 4.4 ± 1.7 bp) and unwinding rate (mk_obs = 396 ± 15 bp s^− 1). These results suggest that the additional kinetic steps with rate constant k_C required for RecBCD to initiate unwinding of blunt-ended and twin (dT)₆-tailed DNA reflect processes needed to engage the RecD motor with the 5′ ssDNA.