Interactions of human O6-alkylguanine-DNA alkyltransferase (AGT) with short single-stranded DNAs

The O6-alkylguanine-DNA alkyltransferase (AGT) repairs O6-alkylguanine and O4-alkylthymine adducts in single-stranded and duplex DNAs. Here we characterize the binding of AGT to single-stranded DNAs ranging in length from 5 to 78 nucleotides (nt). Binding is moderately cooperative (37.9 +/- 3.0 相似文献   

Enzymatic removal of O6-ethylguanine from mitochondrial DNA in rat tissues exposed to N-ethyl-N-nitrosourea in vivo

DNA repair is essential for maintaining the integrity of the genetic material, and a number of DNA repair mechanisms have been fairly well characterized for the nuclear DNA of eukaryotic cells as well as prokaryotes. However, little is known about DNA repair in mitochondria. Using highly sensitive immunoanalytical methods to detect specific DNA alkylation products, we found active removal of O6-ethyl-2'-deoxyguanosine (O6-EtdGuo) from rat liver mitochondrial DNA after pulse-exposure to N-ethyl-N-nitrosourea in vivo. In the kidney, O6-EtdGuo was removed from mitochondrial DNA with moderate efficiency, but nearly no removal was observed from the DNA of brain mitochondria. Among the rat tissues examined, the kinetics of O6-EtdGuo elimination from mitochondrial DNA was very similar to the kinetics of removal from nuclear DNA. O4-Ethyl-2'-deoxythymidine, another premutagenic DNA ethylation product, was stable in both mitochondrial and nuclear DNA of rat liver.     

Thermodynamic investigation of monoclonal antibody alkylated nucleoside interaction as a model for epitope recognition on nucleic acids

The objective of this investigation is examination of the dominant forces that govern complex formation between a series of monoclonal antibodies directed against O6-ethyl-2'-deoxyguanosine. These monoclonal antibodies (coded as ER-6, ER-3, and EM-1) provide the basis for a thermodynamic comparative evaluation of the potentially different forces that stabilize the various monoclonal antibody (mAb) alkylated nucleoside complexes. The binding affinities of ER-6, ER-3, and EM-1 are measured in terms of specific (O6-ethyl-2'-deoxyguanosine, or O6-EtdGuo) and nonspecific (O6-methyl-2'-deoxyguanosine, or O6-MedGuo) antigens, under a variety of experimental conditions, including pH, sodium chloride addition, 1-propanol addition, and temperature, via a nitrocellulose affinity filter assay. The binding isotherms were analyzed via a least-squares routine fit to a two independent binding sites model. The temperature dependence of the van't Hoff enthalpies for the specific O6-EtdGuo interaction ranges from -15.18 to -18.60 kcal mol-1, while for O6-MedGuo the range was extended from -2.72 to -20.66 kcal mol-1. The standard and unitary entropies were negative for those mAb interactions with O6-EtdGuo as well as for ER-6/O6-MedGuo complex formation. However, it was found that the interactions between ER-3 and EM-1 with O6-MedGuo led to decidedly positive entropic values. These results indicate two different dominant forces at work in complex stabilization. The interaction of the three mAb's with their specific antigen, as well as ER-6/O6-MedGuo interaction (nonspecific), may well be controlled by van der Waals type forces, while ER-3 and EM-1 interactions with nonspecific antigen imply formal charge neutralization electrostatics as the dominant force.     

Titin: quantitative mass measurements by scanning transmission electron microscopy and structural implications for the sarcomere matrix of skeletal muscle

Scanning transmission electron microscopy has been used to investigate mass and linear mass density of native titin-2, a large soluble fragment of intact titin, from rabbit skeletal muscle. Dark field images of unstained, freeze-dried titin-2 appeared as either compact globules or looser and larger balls of string. Direct mass measurements indicated that the compact forms have an average mass of 2.40 +/- 0.50 x 10(6) Da. The mass to length ratio, determined from well-spread portions of titin strands (3-5 nm wide) from the ball of string forms, averaged 2.7 +/- 0.9 kDa/nm. Thus a single native intact titin molecule has a calculated contour length of well above approximately 1 micron, sufficient to span unidirectionally between the Z line and M line region in a resting-length sarcomere.     

The vnd/NK-2 homeodomain: thermodynamics of reversible unfolding and DNA binding for wild-type and with residue replacements H52R and H52R/T56W in helix III

The conformational stabilities of the vnd (ventral nervous system defective)/NK-2 homeodomain [HD(wt); residues 1-80 that encompass the 60-residue homeodomain] and those harboring mutations in helix III of the DNA recognition site [HD(H52R) and HD(H52R/T56W)] have been investigated by differential scanning calorimetry (DSC) and ellipticity changes at 222 nm. Thermal unfolding reactions at pH 7.4 are reversible and repeatable in the presence of 50-500 mM NaCl with DeltaC(p) = 0.52 +/- 0.04 kcal K(-1) mol(-1). A substantial stabilization of HD(wt) is produced by 50 mM phosphate or by the addition of 100-500 mM NaCl to 50 mM Hepes, pH 7.4, buffer (from T(m) = 35.5 degrees C to T(m) 43-51 degrees C; DeltaH(vH) congruent with 47 +/- 5 kcal mol(-1)). The order of stability is HD(H52R/T56W) > HD(H52R) > HD(wt), irrespective of the anions present. Progress curves for ellipticity changes at 222 nm as a function of increasing temperature are fitted well by a two-state unfolding model, and the cooperativity of secondary structure changes is greater for mutant homeodomains than for HD(wt) and also is increased by adding 100 mM NaCl to Hepes buffer. A 33% quench of the intrinsic tryptophanyl residue fluorescence of HD(wt) by phosphate binding (K(D)' = 2.6 +/- 0.3 mM phosphate) is reversed approximately 60% by DNA binding. Thermodynamic parameters for vnd/NK-2 homeodomain proteins binding sequence-specific 18 bp DNA have been determined by isothermal titration calorimetry (10-30 degrees C). Values of DeltaC(p) are +0.25, -0.17, and -0.10 +/- 0.04 kcal K(-1) mol(-1) for HD(wt), HD(H52R), and HD(H52R/T56W) binding duplex DNA, respectively. Interactions of homeodomains with DNA are enthalpically controlled at 298 K and pH 7.4 with corresponding DeltaH values of -6.6 +/- 0.5, -10.8 +/- 0.1, and -9.0 +/- 0.6 kcal mol(-1) and DeltaG' values of -11.0 +/- 0.1, -11.0 +/- 0.1, and -11.3 +/- 0.3 kcal mol(-1) with a binding stoichiometry of 1.0 +/- 0.1. Thermodynamic parameters for DNA binding are not predicted from homeodomain structural changes that occur upon complexing to DNA and must reflect also solvent and possibly DNA rearrangements.     

Differential modulation of actin-severing activity of gelsolin by multiple isoforms of cultured rat cell tropomyosin. Potentiation of protective ability of tropomyosins by 83-kDa nonmuscle caldesmon

Multiple isoforms of tropomyosin (TM) of rat cultured cells show differential effects on actin-severing activity of gelsolin. Flow birefringence measurements have revealed that tropomyosin isoforms with high Mr values (high Mr TMs) partially protect actin filaments from fragmentation by gelsolin, while tropomyosins with low Mr values (low Mr TMs) have no significant protection even when the actin filaments have been fully saturated with low Mr TMs. We have also examined effect of nonmuscle caldesmon on the severing activity of gelsolin because 83-kDa nonmuscle caldesmon stimulates actin binding of rat cell TMs (Yamashiro-Matsumura, S., and Matsumura, F. (1988) J. Cell Biol. 106, 1973-1983). While nonmuscle caldesmon alone or low Mr TMs alone show no significant protection against fragmentation by gelsolin, the low Mr TMs coupled with 83-kDa protein are able to protect actin filaments. Further, high Mr TMs together with 83-kDa protein appear to block the severing activity completely. Electron microscopic analyses of length distribution of actin filaments have confirmed the results. The average length of control actin filaments is measured as 1.46 +/- microns, and gelsolin shortens the average length to 0.084 +/- 0.039 micron. Similar short average lengths are obtained when gelsolin severs actin complexed with low Mr TMs (0.080 +/- 0.045 micron) or with nonmuscle caldesmon (0.11 +/- 0.072 micron) while longer average length (0.22 +/- 0.18 micron) is measured in the presence of high Mr TMs. The simultaneous addition of nonmuscle caldesmon makes the average length considerably longer, i.e. 0.61 +/- 0.37 micron in the presence of low Mr TMs and 1.57 +/- 0.97 micron in the presence of high Mr TMs. Furthermore, the actin binding of gelsolin is strongly inhibited by co-addition of high Mr TMs and nonmuscle caldesmon. These results suggest that TM and gelsolin share the same binding site on actin molecules and that the differences in the actin affinities between TMs are related to their abilities of protection against gelsolin.     

DNA condensation by polyamines: a laser light scattering study of structural effects.

Polyamines such as spermidine and spermine are abundant in living cells and are believed to aid in the dense packaging of cellular DNA. DNA condensation is a prerequisite for the transport of gene vectors in living cells. To elucidate the structural features of polyamines governing DNA condensation, we studied the collapse of lambda-DNA by spermine and a series of its homologues, H2N(CH2)3NH(CH2)n=2-12NH(CH2)3NH2 (n = 4 for spermine), using static and dynamic light scattering techniques. All polyamines provoked DNA condensation; however, their efficacy varied with the structural geometry of the polyamine. In 10 mM sodium cacodylate buffer, the EC50 values for DNA condensation were comparable (4 +/- 1 microM) for spermine homologues with n = 4-8, whereas the lower and higher homologues provoked DNA condensation at higher EC50 values. The EC50 values increased with an increase in the monovalent ion (Na+) concentration in the buffer. The slope of a plot of log [EC50(polyamine4+)] against log [Na+] was approximately 1.5 for polyamines with even number values of n, whereas the slope value was approximately 1 for compounds with odd number values of n. Dynamic light scattering measurements showed the presence of compact particles with hydrodynamic radii (Rh) of about 40-50 nm for compounds with n = 3-6. Rh increased with further increase in methylene chain length separating the secondary amino groups of the polyamines (Rh = 60-70 nm for n = 7-10 and >100 nm for n = 11 and 12). Determination of the relative binding affinity of polyamines to DNA using an ethidium bromide displacement assay showed that homologues with n = 2 and 3 as well as those with n > 7 had significantly lower DNA binding affinity compared to spermine and homologues with n = 5 and 6. These data suggest that the chemical structure of isovalent polyamines exerts a profound influence on their ability to recognize and condense DNA, and on the size of the DNA condensates formed in aqueous solution.     

Characteristics of erythrocyte diagnostic agents studied using optical methods

The quantitative characterization of erythrocyte diagnosticums (ED) has been made by optical methods (light microscopy with the use of an image analyzer, model Magiscan 2, and the opacity spectrum technique). The following parameters of ED have been determined: the average of the major axis (5.25 +/- 0.57 micron for ED from Shigella sonnei and 5.53 +/- 0.50 micron for ED from Shigella flexneri), the ratio of semiaxes (p approximately equal to 3), the major axis length distribution, the refractive index (1.076 +/- 0.002). For controlling the concentration of ED the use of the opacity spectrum technique is recommended.     

Changes in cell surface primary cilia and microvilli concurrent with measurements of fluid flow across the rabbit corneal endothelium ex vivo

Primary cilia and microvilli have been reported on the mammalian rabbit corneal endothelium but their relationship to cell function is undefined. Six corneas from healthy 2 kg female albino rabbits were glutaraldehyde-fixed post mortem (15:00 h) or twelve corneal stroma-endothelial preparations incubated at 37 degrees C under an applied hydrostatic pressure of 20 cm H2O for 4 h prior to fixation. The corneal endothelium was assessed by quantitative scanning electron microscopy. Cells fixed immediately post mortem were decorated with small stubby microvilli (average 21 +/- 13/100 micron 2), and only 25% of the cells were decorated with primary cilia having an average length of 2.44 +/- 1.56 microns. Following 4 h ex vivo incubation with a phosphate-buffered Ringer solution, conspicuous microvilli developed to an average density of 40 +/- 19/100 micron 2 and primary cilia were found on 12% of the cells, having on average length of 2.27 +/- 1.38 microns. Following 4 h incubation in a bicarbonate-buffered Ringer solution, small stubby microvilli developed to a density of 49 +/- 18/100 micron 2, and 40% of the cells showed primary cilia with an average length of 4.31 +/- 1.93 microns; the net trans-endothelial fluid flow in the latter set was 60% greater. These studies indicate that the primary cilia on corneal endothelial cells might be responsive to fluid flow, but that mild mechanical and/or chemical stress could also be the cause of the change since the elaboration of primary cilia can be accompanied by microvilli as well.     

Structural and enzymological characterization of the homogeneous deoxyribonucleic acid polymerase from Mycoplasma orale.

We have purified the DNA polymerase from Mycoplasma orale to homogeneity. The protein structure of the enzyme was declined by sodium dodecyl sulfate gel electrophoresis, which revealed a single band of 116 000 daltons that was coincident with the polymerase activity profile in the final step of DNA--cellulose chromatography, and by two-dimensional gel analysis, which demonstrated a single protein species at pI = 6.8 that was congruent with enzyme activity and contained the same 116 000 polypeptide. although severe enzyme aggregation occurs during nondenaturing gel electrophoresis, a monomer species can be resolved with a Mr of 140 000 by the Ferguson plot analysis. Gel filtration and velocity gradient centrifugation yield a Stokes radius of 4.8 nm and a sedimentation coefficient of 5.6 S, respectively, from which Mr values of 106 000--128 000 can be computed. The different size values suggest that the polymerase molecule is asymmetric. The purified enzyme has a specific activity of approximately 6 x 10(5) units/mg of protein and in completely devoid of exodeoxyribonuclease and endodeoxyribonuclease activities, at exclusion limits of 10(-4)--10(-6%) of the polymerase activity. The mechanism of polymerization is moderately processive, with an average of 14 +/- 4 nucleotides incorporated per binding event, and the "effective template length" on activated DNA is approximately 40 nucleotides.     

Conformation of reconstituted mononucleosomes and effect of linker histone H1 binding studied by scanning force microscopy

The conformation of mononucleosome complexes reconstituted with recombinant core histones on a 614-basepair-long DNA fragment containing the Xenopus borealis 5S rRNA nucleosome positioning sequence was studied by scanning/atomic force microscopy in the absence or presence of linker histone H1. Imaging without prior fixation was conducted with air-dried samples and with mononucleosomes that were injected directly into the scanning force microscopy fluid cell and visualized in buffer. From a quantitative analysis of approximately 1,700 complexes, the following results were obtained: i), In the absence of H1, a preferred location of the nucleosome at the X. borealis 5S rRNA sequence in the center of the DNA was detected. From the distribution of nucleosome positions, an energy difference of binding to the 5S rRNA sequence of DeltaDeltaG approximately 3 kcal mol(-1) as compared to a random sequence was estimated. Upon addition of H1, a significantly reduced preference of nucleosome binding to this sequence was observed. ii), The measured entry-exit angles of the DNA at the nucleosome in the absence of H1 showed two maxima at 81 +/- 29 degrees and 136 +/- 18 degrees (air-dried samples), and 78 +/- 25 degrees and 137 +/- 25 degrees (samples imaged in buffer solution). In the presence of H1, the species with the smaller entry-exit angle was stabilized, yielding average values of 88 +/- 34 degrees for complexes in air and 85 +/- 10 degrees in buffer solution. iii), The apparent contour length of the nucleosome complexes was shortened by 34 +/- 13 nm as compared to the free DNA due to wrapping of the DNA around the histone octamer complex. Considering an 11 nm diameter of the nucleosome core complex, this corresponds to a total of 145 +/- 34 basepairs that are wound around the nucleosome.     

Salt-dependent DNA superhelix diameter studied by small angle neutron scattering measurements and Monte Carlo simulations.

Using small angle neutron scattering we have measured the static form factor of two different superhelical DNAs, p1868 (1868 bp) and pUC18 (2686 bp), in dilute aqueous solution at salt concentrations between 0 and 1.5 M Na+ in 10 mM Tris at 0% and 100% D2O. For both DNA molecules, the theoretical static form factor was also calculated from an ensemble of Monte Carlo configurations generated by a previously described model. Simulated and measured form factors of both DNAs showed the same behavior between 10 and 100 mM salt concentration: An undulation in the scattering curve at a momentum transfer q = 0.5 nm-1 present at lower concentration disappears above 100 mM. The position of the undulation corresponds to a distance of approximately 10-20 nm. This indicated a change in the DNA superhelix diameter, as the undulation is not present in the scattering curve of the relaxed DNA. From the measured scattering curves of superhelical DNA we estimated the superhelix diameter as a function of Na+ concentration by a quantitative comparison with the scattering curve of relaxed DNA. The ratio of the scattering curves of superhelical and relaxed DNA is very similar to the form factor of a pair of point scatterers. We concluded that the distance of this pair corresponds to the interstrand separation in the superhelix. The computed superhelix diameter of 16.0 +/- 0.9 nm at 10 mM decreased to 9.0 +/- 0.7 nm at 100 mM salt concentration. Measured and simulated scattering curves agreed almost quantitatively, therefore we also calculated the superhelix diameter from the simulated conformations. It decreased from 18.0 +/- 1.5 nm at 10 mM to 9.4 +/- 1.5 nm at 100 mM salt concentration. This value did not significantly change to lower values at higher Na+ concentration, in agreement with results obtained by electron microscopy, scanning force microscopy imaging in aqueous solution, and recent MC simulations, but in contrast to the observation of a lateral collapse of the DNA superhelix as indicated by cryo-electron microscopy studies.     

Testosterone-induced development of the rat levator ani muscle

The perinatal development of the levator ani (LA) muscle in male and female rats was investigated by measuring the total number of muscle units (MU) (i.e., mononucleate cells, clustered or independent myotubes, and muscle fibers) in transverse semithin sections of the entire muscle and the MU cross-sectional area in 22-day-old fetuses (F22), 1-day-old (D1 = day of birth), 3-day-old (D3), and 6-day-old (D6) newborns. Male muscle contained 350 +/- 64 MU on F22, twice that of the female. The number of MU increased markedly in males from F22, but changed little in females; the number of MU in males was 760% that of females on D6. The MU cross-sectional area was greater in males on F22 (120.8 micron(s)2 +/- 7.5) and D1 (155.2 micron(s)2 +/- 64.8) than in females (F22: 89.2 micron(s) +/- 14.2, D1: 64.1 micron(s)2 +/- 19.7) and dropped to about 30 X micron(s)2 in both sexes on D6. Female rats given a single injection of testosterone propionate (TP) before D7 showed a significant increase in the number of fibers, but no increase in cross-sectional area. TP given after D7 had no effect on the fiber number, but increased the average cross-sectional area. The increase in fiber number induced by postnatal TP treatment was a permanent effect, still quantifiable in 15-month-old females. We conclude that the sexual dimorphism of the rat LA muscle is principally due to a dramatic increase in the MU number in male muscles during the perinatal period, rather than to involution of the fibers in female muscles as it is widely accepted. This increase seems to be, at least partly, under the control of testosterone.     

Human muscle sarcoplasmic reticulum function during submaximal exercise in normoxia and hypoxia.

In this study, the response of the sarcoplasmic reticulum (SR) to prolonged exercise, performed in normoxia (inspired O(2) fraction = 0.21) and hypoxia (inspired O(2) fraction = 0.14) was studied in homogenates prepared from the vastus lateralis muscle in 10 untrained men (peak O(2) consumption = 3.09 +/- 0.25 l/min). In normoxia, performed at 48 +/- 2.2% peak O(2) consumption, maximal Ca(2+)-dependent ATPase activity was reduced by approximately 25% at 30 min of exercise compared with rest (168 +/- 10 vs. 126 +/- 8 micromol.g protein(-1) x min(-1)), with no further reductions observed at 90 min (129 +/- 6 micromol x g protein(-1) x min(-1)). No changes were observed in the Hill coefficient or in the Ca(2+) concentration at half-maximal activity. The reduction in maximal Ca(2+)-dependent ATPase activity at 30 min of exercise was accompanied by oxalate-dependent reductions (P < 0.05) in Ca(2+) uptake by approximately 20% (370 +/- 22 vs. 298 +/- 25 micromol x g protein(-1) x min(-1)). Ca(2+) release, induced by 4-chloro-m-cresol and assessed into fast and slow phases, was decreased (P < 0.05) by approximately 16 and approximately 32%, respectively, by 90 min of exercise. No differences were found between normoxia and hypoxia for any of the SR properties examined. It is concluded that the disturbances induced in SR Ca(2+) cycling with prolonged moderate-intensity exercise in human muscle during normoxia are not modified when the exercise is performed in hypoxia.     

Levels of O6-methylguanine acceptor protein in extracts of human breast tumor tissues.

We have measured the abilities of extracts of tissues from human breast tumors to demethylate adducts of O6-meG in exogenous DNA by transfer of the methyl group to an acceptor protein. The results have shown that all 21 specimens examined (including 5 non-neoplastic, 11 malignant tumors and 5 benign growth) contained significant amounts of O6-meG acceptor activity, removing on average 221.1 +/- 2.1 (SEM) fmol O6-meG per mg protein or 10.07 +/- 0.98 (SEM) fmol O6-megG per microgram DNA in the extracts. There were also wide interindividual variations, which were not age-dependent, and there were no significant differences between the non-neoplastic and neoplastic tissues obtained from individuals with benign or with malignant disease. It was estimated that the average number of O6-meG acceptor molecules per cell in normal human breast tissues was calculated as 46,000 +/- 7000 (SEM).     

Energetics of DNA twisting. II. Topoisomer analysis

A gel electrophoresis method has been developed for resolving small (approximately equal to 250 bp DNA topoisomers. In this size range only one major topoisomer band is observed, except for ligase closure conditions in which the probabilities are nearly equal for circularization by untwisting and overtwisting the corresponding linear DNA. The two probabilities are nearly equal when delta Tw is close to 0.5, if the mean helical twist of the linear DNA is n + delta Tw, where n is an integer and delta Tw is the fractional twist. We determine delta Tw of the linear DNA in standard conditions (20 degrees C, no ethidium) by titration experiments in which delta Tw is varied at the time of ligase closure, either by changing temperature or ethidium concentration. The endpoint (delta Tw = 0.5) is found when the two topoisomers formed by untwisting and overtwisting are present at equal concentrations. This analysis assumes that the net writhe is zero and the DNA helix is isotropically bendable. The results confirm the analysis of cyclization probabilities given in the preceding paper: delta Tw = 0 at the two maxima in the curve of j-factor versus DNA length and delta Tw = 0.5 at the minimum. Consequently, we can determine the DNA lengths at which Tw takes on integral values and use them to measure precisely the average helix repeat. From the difference between the delta Tw values of DNAs with 237 and 247 bp, we obtain an approximate value for the helix repeat of h = 10.4 +/- 0.1 bp/turn, in good agreement with earlier values found by the band-shift and nuclease-cutting methods. The twist is integral at 250.8 +/- 0.4 bp and from h = 10.4 +/- 0.1 we find n = 24; then 250.8/24 gives h = 10.45 +/- 0.02 bp/turn. The mean linking number (Lk) changes in a stepwise manner as delta Tw is varied for 250 bp DNAs. This result is expected when the free energy of twisting half a turn becomes large compared to thermal fluctuations. In these experiments, it is possible to obtain the mean Tw value from the mean Lk value only when delta Tw = 0.5, and consequently the mean Lk value is not simply related to DNA length for 250 bp DNAs except when delta Tw = 0.5.(ABSTRACT TRUNCATED AT 400 WORDS)     

An immobilized fork as a termination of replication intermediate in Bacillus subtilis

The structure of a DNA intermediate associated with termination of chromosome replication in Bacillus subtilis and derived from a unique BamHI 24.8 X 10(3) base-pair (bp) region of the chromosome has been investigated. The intermediate has properties expected for a forked structure. Gel electrophoresis followed by Southern transfer and hybridization to cloned DNA has shown it to comprise single strands of 15.4 X 10(3) bp and 24.8 X 10(3) bp, in approximately equimolar amounts. After purification away from the bulk of chromosomal DNA, electron microscopy of the intermediate established that 15% of the DNA was present as branched molecules and a significant proportion (11 of 31) of these contained two arms of matching length. The average dimensions (best estimates) of this unique class of Y-shaped molecule were 9.5(+/- 0.3) X 10(3), 15.1(+/- 0.4) X 10(3) and 24.6 24.6(+/- 0.6) X 10(3) bp for the stem, arms and end-to-end length, respectively. These values are consistent with the single strand composition of the intermediate as found. Furthermore, hybridization of the single strands to DNA from known locations within the BamHI 24.8 X 10(3) bp region has established the orientation of the forked intermediate relative to the genetic map. The intermediate presumably reflects the immobilization of the clockwise replication fork within the 24.8 X 10(3) bp region, at a location approximately 15.4 X 10(3) bp from the right end.     

Sequestering of p53 into DNA-protein filaments revealed by electron microscopy

Using electron microscopy, we analyzed the interaction of bacterially expressed full-length p53, p53(1-393), and its C-terminal fragment, p53(320-393), with long (approximately 3000 bp) dsDNA in linear and supercoiled (|DeltaLk| approximately 4-6) forms containing or lacking the p53 recognition sequence (p53CON). The main structural feature of the complexes formed by either protein was a DNA-protein filament, in which two DNA duplexes are linked (synapsed) via bound protein tetramers. The efficiency of the synapse, reflected in its length and the fraction of molecules exhibiting DNA-protein filaments, was significantly modulated by the molecular form of the protein and the topological state of the DNA. With linear DNA, the synapse yield promoted by the C-terminus fragment was very low, but the full-length protein was effective in linking noncontiguous duplexes, leading to the formation of intramolecular loops constrained at their bases by short regions of synapsed DNA duplexes. When the linear DNA contained p53CON, regions of preferential sequence, i.e., encompassing p53CON and probably p53CON-like sequences, were predominantly synapsed, indicating a sequence specificity of the p53 core domain. With scDNA, the synapse yield was significantly higher compared to the linear counterparts and was weakly dependent on the sign of superhelicity and presence or absence of p53CON. However, the full-length protein was more effective in promoting DNA synapses compared to the C-terminal fragment. The overall structure of the DNA-protein filaments was apparently similar for either protein form, although the apparent width differed slightly (approximately 7-9 nm and approximately 10-12 nm for p53(320-393) and p53(1-393), respectively). No distortion of the DNA helices involved in the synapse was found. We conclude that the structural similarity of DNA-protein filaments observed for both proteins is attributable mainly to the C-terminus, and that the yield is dictated by the specific and possibly nonspecific interactions of the core domain in combination with DNA topology. Possible implications for the sequestering of p53 in DNA-protein filaments are discussed.