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1.
Interaction between polylysine and DNA's of varied G + C contents was studied using thermal denaturation and circular dichroism (CD). For each complex there is one melting band at a lower temperature tm, corresponding to the helix–coil transition of free base pairs, and another band at a higher temperature tm, corresponding to the transition of polylysine-bound base pairs. For free base pairs, with natural DNA's and poly(dA-dT) a linear relation is observed between the tm and the G + C content of the particular DNA used. This is not true with poly(dG)·poly(dC), which has a tm about 20°C lower than the extrapolated value for DNA of 100% G + C. For polylysine-bound base pairs, a linear relation is also observed between the tm and the G + C content of natural DNA's but neither poly(dA-dT) nor poly(dG)·poly(dC) complexes follow this relationship. The dependence of melting temperature on composition, expressed as dtm/dXG·C, where XG·C is the fraction of G·C pairs, is 60°C for free base pairs and only 21°C for polylysine-bound base pairs. This reduction in compositional dependence of Tm is similar to that observed for pure DNA in high ionic strength. Although the tm of polylysine-poly(dA-dT) is 9°C lower than the extrapolated value for 0% G + C in EDTA buffer, it is independent of ionic strength in the medium and is equal to the tm0 extrapolated from the linear plot of tm against log Na+. There is also a noticeable similarity in the CD spectra of polylysine· and polyarginine·DNA complexes, except for complexes with poly(dA-dT). The calculated CD spectrum of polylysine-bound poly(dA-dT) is substantially different from that of polyarginine-bound poly(dA-dT).  相似文献   

2.
The ground-state tautomerization of the G·C Watson–Crick base pair by the double proton transfer (DPT) was comprehensively studied in vacuo and in the continuum with a low dielectric constant (??=?4), corresponding to a hydrophobic interface of protein–nucleic acid interactions, using DFT and MP2 levels of quantum-mechanical (QM) theory and quantum theory “Atoms in molecules” (QTAIM). Based on the sweeps of the electron-topological, geometric, polar, and energetic parameters, which describe the course of the G·C???G?·C? tautomerization (mutagenic tautomers of the G and C bases are marked with an asterisk) through the DPT along the intrinsic reaction coordinate (IRC), it was proved that it is, strictly speaking, a concerted asynchronous process both at the DFT and MP2 levels of theory, in which protons move with a small time gap in vacuum, while this time delay noticeably increases in the continuum with ??=?4. It was demonstrated using the conductor-like polarizable continuum model (CPCM) that the continuum with ??=?4 does not qualitatively affect the course of the tautomerization reaction. The DPT in the G·C Watson–Crick base pair occurs without any intermediates both in vacuum and in the continuum with ??=?4 at the DFT/MP2 levels of theory. The nine key points along the IRC of the G·C base pair tautomerization, which could be considered as electron-topological “fingerprints” of a concerted asynchronous process of the tautomerization via the DPT, have been identified and fully characterized. These key points have been used to define the reactant, transition state, and product regions of the DPT reaction in the G·C base pair. Analysis of the energetic characteristics of the H-bonds allows us to arrive at a definite conclusion that the middle N1H?N3/N3H?N1 and the lower N2H?O2/N2H?O2 parallel H-bonds in the G·C/G?·C? base pairs, respectively, are anticooperative, that is, the strengthening of the middle H-bond is accompanied by the weakening of the lower H-bond. At that point, the upper N4H?O6 and O6H?N4 H-bonds in the G·C and G?·C? base pairs, respectively, remain constant at the changes of the middle and the lower H-bonds at the beginning and at the ending of the G·C???G?·C? tautomerization. Aiming to answer the question posed in the title of the article, we established that the G?·C? Löwdin’s base pair satisfies all the requirements necessary to cause point mutations in DNA except its lifetime, which is much less than the period of time required for the replication machinery to forcibly dissociate a base pair into the monomers (several ns) during DNA replication. So, from the physicochemical point of view, the G?·C? Löwdin’s base pair cannot be considered as a source of point mutations arising during DNA replication.  相似文献   

3.
Non-suppressible addition frameshift in Salmonella   总被引:2,自引:0,他引:2  
A frameshift mutation in the histidinol dehydrogenase gene of Salmonella was isolated after induction with the intercalating agent, ICR-1914. Reversion of the frameshift, 2578, is strongly enhanced by ICR and by the alkylating agent N-methyl-?-nitro-N-nitrosoguanidine. In all cases previously examined, frame-shifts with this reversion profile have proven to be +1 types, most likely containing an extra G·C pair in a DNA repeat of G·C pairs. Most are suppressible by external suppressors, which appear to translate the +1 site on mRNA as proline or glycine. Frameshift 2578, however, is one of a small minority which, while reverted by ICR-191 and N-methyl-?-nitro-N-nitrosoguanidine, does not appear to be suppressible by external suppressors. Sequence studies of revertant histidinol dehydrogenase suggest that 2578 is an addition of one or two G·C pairs in a DNA repeat of G·C pairs. This addition, however, produces mRNA quadruplets which are in an incorrect phase for suppressor translation.  相似文献   

4.
Zinc fingers, 30-residue peptides anchored on Zn(II) coordinated to pairs of cysteines and histidines, recognize DNA triplets and, as tandem modules, effect sequence read out. The focus of zinc finger-DNA interaction studies thus far has been to probe the nature of the binding of the 12-residue recognition element of the finger with DNA code bases. To understand the possible role of the Zn(II) ligand and to assess its own DNA interaction profile, [(CH)2Zn] (C: cysteine; H: histidine; Figure 1) was constructed from bis-t Boc-cystinyl-di-His-OMe via thiol-disulfide exchange, Zn(II) complexation, and deprotection. [(CH)2Zn] binds with polyd- (G·C)·polyd(G·C) with association constants—1.8 × 107 M−1 (specific DNA-phosphate) and 3.3 × 103 M−1 (nonspecific DNA-phosphate); perturbs its B-DNA profile; and enhances the Tm from 62.5 to 70.15°C in a concentration-independent manner, with an ideal reversal profile on cooling, not observed in the DNA alone; releases polyd(G·C)·polyd(G·C)-bound ethidium bromide; enhances the fluorescence of polyd(G·C)·polyd(G·C)-bound ethidium bromide at low concentrations; and quenches it at higher ranges. [(CH)2Zn] also binds to d(ACGCTGGGCGT), the sequence associated with Zif-268, 3-finger binding site. Such interactions were not seen in parallel studies with (a) polyd(A·T)·polyd(A·T) and [(CH)2Zn] and (b) {[C′H2] (C′: cystine; H: histidine; the direct metal-free precursor of [(CH)2Zn]}, ionic zinc nitrate, and covalent zinc acetyl acetonate Zn(AcAc)2, with poly[d(G·C)·polyd(G·C)]. The results are rationalized on the basis of two types of association between [(CH)2Zn] and polyd(G·C)·polyd(G·C), a nonspecific recognition of the sugar phosphate backbone, by an imidazole of [(CH)2Zn] and a specific one involving the amino group of [(CH)2Zn] and the guanine base of DNA. Control experiments show that the latter greatly promotes DNA recognition. The possibility for such specific interactions with relatively small structures of the type [(CH)2Zn] would be of use in the design of DNA recognition elements and also provide an explanation for the experimentally found variation in the placement of the zinc finger docking unit around the major groove of DNA. © 1997 John Wiley & Sons, Inc.  相似文献   

5.
The affinity of quinacrine for native DNA has been determined from fluorescence measurements and equilibrium dialysis in Tris-HC10.05 m, NaCl0.1 m, EDTA 10?3m, pH 7.5. When considering M. lysodeiktikus, E. coli calf thymus and C. perfringens the affinities of DNA for quaniactive have been found to change by a factor of two and the fluorescence intensities to change by a factor of 25. The varying affinities and fluoroescence intensities of bound quinacrine are attributed to heterogeneous binding. For all DNAs we have assumed that there exist three classes of intercalation sites: I, A·T-A·T; 2, G·C-G·C; and 3, A·T-G·C, assuming that base pair ordering is less relevant than base composition of sites. By fitting the affinities of native DNAs with this model it was found that quinacrine binds to site 2 three times more strongly than it does to site 1. When flucrescence intensity is studied, triplets of A·T pairs appear to be responsible for the high quantum yield of A·T rich DNA whereas the quenching properties of a G·C base pair adjacent to an intercalated quinacrine are well known.  相似文献   

6.
Based on equilibrium binding studies, as well as on kinetic investigations, two types of interactions of Cu2+ ions with native DNA at low ionic strength could be characterized, namely, a nondenaturing and a denaturing complex formation. During a fast nondenaturing complex formation at low relative ligand concentrations and at low temperatures, different binding sites at the DNA bases become occupied by the metal ions. This type of interaction includes chelate formation of Cu2+ ions with atoms N(7) of purine bases and the oxygens of the corresponding phosphate groups, chelation between atoms N(7) and O of C(6) of the guanine bases, as well as the formation of specific intestrand crosslink complexes at adjacent G°C pairs of the sequence dGpC. CD spectra of the resulting nondenatured complex (DNA–Cu2+)nat may be interpreted in terms of a conformational change of DNA from the B-form to a C-like form on ligand binding. A slow cooperative denaturing complex formation occurs at increased copper concentrations and/or at increased temperatures. The uv absorption and CD spectra of the resulting complex, (DNA–Cu2+)denat, indicate DNA denaturation during this type of interaction. Such a conclusion is confirmed by microcalorimetric measurements, which show that the reaction consumes nearly the same amount of heat as acid denaturation of DNA. From these and the kinetic results, the following mechanism for the denaturing action of the ligands is suggested: binding of Cu2+ ions to atoms N(3) of the cytosine bases takes place when the cytosines come to the outside of the double helix as a result of statistical fluctuations. After the completion of the binding process, the bases cannot return to their initial positions, and thus local denaturation at the G·C pairs is brought about. The probability of the necessary fluctuations occurring is increased by chelation of Cu2+ ions between atoms N(7) and O of C(6) of the guanine bases during nondenaturing complex formation, which loosens one of the hydrogen bonds within the G·C pairs, as well as by raising the temperature. The implications of the new binding model, which comprises both the sequence-specific interstand crosslinks and the described mechanism of denaturing complex formation, are discussed and some predictions are made. The model is also used to explain the different renaturation properties of the denatured complexes of Cu2+, Cd2+, and Zn2+ ions with DNA. In temperature-jump experiments with the nondenatured complex (DNA–Cu2+)nat, a specific kinetic effect is observed, namely, the appearance of a lag in the response to the perturbation. The resulting sigmoidal shape of the kinetic curves is considered to be a consequence of the necessity of disrupting a certain number of the crosslinks existing in the nondenatured complex before the local unwinding of the binding regions (a main step of denaturing complex formation) may proceed.  相似文献   

7.
Mithramycin (MTR), an aureolic acid group of antitumor antibiotic is used for the treatment of several types of tumors. We have reported here the association of MTR with an essential micronutrient, manganese (Mn2+). Spectroscopic methods have been used to characterize and understand the kinetics and mechanism of complex formation between them. MTR forms a single type of complex with Mn2+ in the mole ratio of 2:1 [MTR: Mn2+] via a two step kinetic process. Circular dichroism (CD) spectroscopic study indicates that the complex [(MTR)2 Mn2+] has a right-handed twist conformation similar in structure with the complexes reported for Mg2+ and Zn2+. This conformation allows binding via minor groove of DNA with (G, C) base preference during the interaction with double-stranded B-DNA. Using absorbance, fluorescence, and CD spectroscopy we have shown that [(MTR)2 Mn2+] complex binds to double-stranded DNA with an apparent dissociation constant of 32?μM and binding site size of 0.2 (drug/nucleotide). It binds to chicken liver chromatin with apparent dissociation constant value 298?μM. Presence of histone proteins in chromatin inhibits the accessibility of the complex for chromosomal DNA. We have also shown that MTR binds to Mn2+ containing metalloenzyme manganese superoxide dismutase from Escherichia coli.  相似文献   

8.
We have measured the circular dichroism (CD) and absorption properties of poly[r(G-U)] and poly [d(G-T)] over a wide range of Na+ concentrations and temperatures. We find evidence for self-complexed forms of these polymers at lower temperatures and/or higher Na+ concentrations than generally needed for double-strand formation in other DNA and RNA polymers. These self-complexes could be composed of double-stranded regions with weak G·U or G·T base pairs.  相似文献   

9.
C Zimmer  G Luck  H Triebel 《Biopolymers》1974,13(3):425-453
The effects of metal ions of the first-row transition and of alkaline earth metals on the DNA helix conformation have been studied by uv difference spectra, circular dichroism, and sedimentation measurements. At low ionic strength (10?3 M NaClO4) DNA shows a maximum in the difference absorption spectra in the presence of Zn2+, Mn2+, Co2+, Cd2+, and Ni2+ but not with Mg2+ or Ca2+. The amplitude of this maximum is dependent on GC content as revealed by detailed studies of the DNA-Zn2+ complex of eight different DNA's. Pronounced changes also occur in the CD spectra of DNA transition metal complexes. A transition appears up to a total ratio of approximately 1 Zn2+ per DNA phosphate at 10?3 M NaClO4; then no further change was observed up to high concentrations. The characteristic CD changes are strongly dependent on the double-helical structure of DNA and on the GC content of DNA. Differences were also observed in hydrodynamic properties of DNA metal complexes as revealed by the greater increase of the sedimentation coefficient of native DNA in the presence of transition metal ions. Spectrophotometric acid titration experiments and CD measurements at acidic pH clearly indicate the suppression of protonation of GC base-pair regions on the addition of transition metal ions to DNA. Similar effects were not observed with DNA complexes with alkaline earth metal ions such as Mg2+ or Ca2+. The data are interpreted in terms of a preferential interaction of Zn2+ and of other transition metal ions with GC sites by chelation to the N-7 of guanine and to the phosphate residue. The binding of Zn2+ to DNA disappears between 0.5 M and 1 M NaClO4, but complex formation with DNA is observable again in the presence of highly concentrated solutions of NaClO4 (3?7.2 M NaClO4) or at 0.5 to 2 M Mn2+. At relatively high cation concentration Mg2+ is also effective in changing the DNA comformation. These structural alterations probably result from both the shielding of negatively charged phosphate groups and the breakdown of the water structure along the DNA helix. Differential effects in CD are also observed between Mn2+, Zn2+ on one hand and Mg2+ on the other hand under these conditions. The greater sensitivity of the double-helical conformation of DNA to the action of transition metal ions is due to the affinity of the latter to electron donating sites of the bases resulting from the d electronic configuration of the metal ions. An order of the relative phosphate binding ability to base-site binding ability in native DNA is obtained as follows: Mg2+, Ba2+, < Ca2+ < Fe2+, Ni2+, Co2+ < Mn2+, Zn2+ < Cd2+ < Cu2+. The metal-ion induced conformational changes of the DNA are explained by alternation of the winding angle between base pairs as occurs in the transition from B to C conformation. These findings are used for a tentative molecular interpretation of some effects of Zn2+ and Mn2+ in DNA synthesis reported in the literature.  相似文献   

10.
This study provides the first accurate investigation of the tautomerization of the biologically important guanine*·thymine (G*·T) DNA base mispair with Watson–Crick geometry, involving the enol mutagenic tautomer of the G and the keto tautomer of the T, into the G·T* mispair (?G?=?.99?kcal?mol?1, population?=?15.8% obtained at the MP2 level of quantum-mechanical theory in the continuum with ε?=?4), formed by the keto tautomer of the G and the enol mutagenic tautomer of the T base, using DFT and MP2 methods in vacuum and in the weakly polar medium (ε?=?4), characteristic for the hydrophobic interfaces of specific protein–nucleic acid interactions. We were first able to show that the G*·T?G·T* tautomerization occurs through the asynchronous concerted double proton transfer along two antiparallel O6H···O4 and N1···HN3 H-bonds and is assisted by the third N2H···O2 H-bond, that exists along the entire reaction pathway. The obtained results indicate that the G·T* base mispair is stable from the thermodynamic point of view complex, while it is dynamically unstable structure in vacuum and dynamically stable structure in the continuum with ε?=?4 with lifetime of 6.4·10?12?s, that, on the one side, makes it possible to develop all six low-frequency intermolecular vibrations, but, on the other side, it is by three orders less than the time (several ns) required for the replication machinery to forcibly dissociate a base pair into the monomers during DNA replication. One of the more significant findings to emerge from this study is that the short-lived G·T* base mispair, which electronic interaction energy between the bases (?23.76?kcal?mol?1) exceeds the analogical value for the G·C Watson–Crick nucleobase pair (?20.38?kcal?mol?1), “escapes from the hands” of the DNA replication machinery by fast transforming into the G*·T mismatch playing an indirect role of its supplier during the DNA replication. So, exactly the G*·T mismatch was established to play the crucial role in the spontaneous point mutagenesis.  相似文献   

11.
K J Miller 《Biopolymers》1979,18(4):959-980
An algorithm is developed that enables the routine determination of backbone conformations of nucleic acids. All atomic positions including hydrogen are specified in accord with experimental bond lengths and angles but with theoretically determined conformational angles. For two Watson-Crick base pairs at a separation of 3.38 Å, and perpendicular to a common helical axis, minimum energy configurations are found for all 10 combinations at helical angles of α ~ 36°–38°, corresponding to the B-DNA structure with C(2′)-endo sugar puckers. Backbone configurations exist only within the range 35.5° ? α ? 42°, which suggests the origin of the 10-fold helix. Calculated stacking energies for the B-DNA structure increases for each of the clustered groups of base pairs: G·C with G·C, G·C with A·T, and A·T with A·T, and they are in approximate agreement with experimental observations. The counter-clockwise helix is examined, and physically meaningful structures are found only when the helical axes of successive base pairs are disjointed.  相似文献   

12.
Error-prone polymerase chain reactions (epPCRs) are often used to introduce mutations in random mutagenesis, which has been used as a tool in protein engineering. Here, we developed a new method of epPCR using heavy water as a solvent instead of normal water (H2O). Rhodopsin cDNA of the Ayu fish (Plecoglossus altivelis) was used as a template and was amplified using five different conditions: (A) 100% H2O with no Mn2+, (B) 100% H2O/0.6 mM Mn2+, (C) 99% D2O with no Mn2+, (D) 99% D2O/0.6 mM Mn2+ and (E) 99% H218O with no Mn2+. The 13,960 (for each of the conditions A to D) and 33,504 (for condition E) base pairs were sequenced. A maximum error rate of 1.8 × 10−3 errors/bp was detected in condition D, without any particular hot-spot mutations. A high preference for AT → GC transitions was observed in condition D, whereas a high preference for transitions over transversions was observed in condition C. All of the mutations observed in condition E were transversions. When conditions A and C were applied to another template, the honeybee actin gene, the results were comparable to those for Ayu rhodopsin. Based on these results, the use of heavy water, instead of H2O, as a solvent for epPCR can introduce random mutations without positional bias, template dependency or decreased yield. Our new epPCR method, and possibly combining the use of D2O and H218O, may be a powerful random mutagenesis technique.  相似文献   

13.
A S Benight  R M Wartell 《Biopolymers》1983,22(5):1409-1425
Theoretical melting curves were calculated for four DNA restriction fragments, 157–257 base pairs (bp), and a series of hypothetical block DNAs with sequences d(C2xAxC2x). d(C2xTxG2x), 5 ? x ? 40. These DNAs provided a mixture of A·T/G·C sequence distributions with which to investigate the effects of parameters and base-pair changes on the melting of short DNAs. The sensitivity of DNA melting curves to changes in internal loop melting parameters σ and κ was examined. As Expected, theoretical melting curves of short DNAs with a quasirandom base-pair sequence vary little with changes in internal loop parameters. End melting dominates the transition behaviour of these moleucles. This was also observed for the block DNAs up to x = 22. Beyond this length, melting curves are highly sensitive to the internal loop parameters. Sensitivity is also predicted for a 157-bp fragment with a block distribution of A·T and G·C pairs. These results indicate that accurate evaluation of internal loop parameters is possible with short DNAs (100–200 bp) containing a G·C/A·T/G·C block distribution with at least 22 bp in each block. Duplex-to-single-strands dissociation parameters were reevaluated form experimental melting curve data of eight DNA fragments using a least squares fit approach. This analysis confirmed parameter values previously found with a simplified dissociation model. A Priori predictions are made on the effects of base-pair changes on the melting curves of three characterized DNA restriction fragments. Single base-pair changes are predicted to induce small but measurable changes in the melting curves. The characteristics of the altered melting curves depend on the location of the base-pair change.  相似文献   

14.
Five new structures of the Q138F HincII enzyme bound to a total of three different DNA sequences and three different metal ions (Ca2+, Mg2+, and Mn2+) are presented. While previous structures were produced from soaking Ca2+ into preformed Q138F HincII/DNA crystals, the new structures are derived from cocrystallization with Ca2+, Mg2+, or Mn2+. The Mn2+-bound structure provides the first view of a product complex of Q138F HincII with cleaved DNA. Binding studies and a crystal structure show how Ca2+ allows trapping of a Q138F HincII complex with noncognate DNA in a catalytically incompetent conformation. Many Q138F HincII/DNA structures show asymmetry, despite the binding of a symmetric substrate by a symmetric enzyme. The various complexes are fit into a model describing the different conformations of the DNA-bound enzyme and show how DNA conformational energetics determine DNA-cleavage rates by the Q138F HincII enzyme.  相似文献   

15.
R L Ornstein  J R Fresco 《Biopolymers》1983,22(8):1979-2000
Tm values for 20 DNA duplexes with different repeating base sequences provided the data base for developing a rational and relatively simple methodology for computing apparent enthalpies for the helix → coil transitions of DNA helices, ΔH calc. The computational variables and their range of acceptable values were selected on the basis of physically plausible arguments. Over 350,000 different combinations of the variables were tested for degree of fit. It was therby possible to find a combination giving a high degree of linear fit between Tm and ΔH calc (correlation coefficient, 0.99), with Tm values deviating (on average) from the regression line by only ±2.17°C. Most of this uncertainty is attributed to experimental limitations, although computational approximations also contribute. With ΔH calc for the melting of each of the unique complementary dinucleotide fragments computed by the method developed, it is possible to estimate Tm and (relative) ΔH calc reliable for the melting of any particular DNA [with base pairs G(I)·C and A·T] given only its base sequence. The ΔHcalc values for the complementary dinucleotide fragments, together with statistical considerations, make it apparent that Tm of DNAs with repeating base sequence show only an approximate linear dependence on G·C content because A·T and G·G pairs do not contribute to helix stability independently of the base-pair sequence in which they occur. In fact, the nearestneighbor stacking interactions are so significant that certain complementary dinucleotide fragment sequences with 0,50, and 100% G·C content have the same stability.  相似文献   

16.
31P-nmr has been used to investigate the specific interaction of three divalent metal ions, Mg2+, Mn2+, and Co+2, with the phosphate groups of DNA. Mg2+ is found to have no significant effect on any of the 31P-nmr parameters (chemical shift, line-width, T1, T2, and NOE) over a concentration range extending from 20 to 160 mM. The two paramagnetic ions, Mn2+ and Co2+, on the other hand, significantly change the 31P relaxation rates even at very low levels. From an analysis of the paramagnetic contributions to the spin–lattice and spin–spin relaxation rates, the effective internuclear metal–phosphorus distances are found to be 4.5 ± 0.5 and 4.1 ± 0.5 Å for Mn2+ and Co2+, respectively, corresponding to only 15 ± 5% of the total bound Mn2+ and Co2+ being directly coordinated to the phosphate groups (inner-sphere complexes). This result is independent of any assumptions regarding the location of the remaining metal ions which may be bound either as outer-sphere complexes relative to the phosphate groups or elsewhere on the DNA, possibly to the bases. Studies of the temperature effects on the 31P relaxation rates of DNA in the absence and presence of Mn2+ and Co2+ yielded kinetic and thermodynamic parameters which characterize the association and dissociation of the metal ions from the phosphate groups. A two-step model was used in the analysis of the kinetic data. The lifetimes of the inner-sphere complexes are 3 × 10?7 and 1.4 × 10?5 s for Mn2+ and Co2+, respectively. The rates of formation of the inner-sphere complexes with the phosphate are found to be about two orders of magnitude slower than the rate of the exchange of the water of hydration of the metal ions, suggesting that expulsion of water is not the rate-determining step in the formation of the inner-sphere complexes. Competition experiments demonstrate that the binding of Mg2+ ions is 3–4 times weaker than the binding of either Mn2+ or Co2+. Since the contribution from direct phosphate coordination to the total binding strength of these metal ion complexes is small (~15%), the higher binding strength of Mn2+ and Co2+ may be attributed either to base binding or to formation of stronger outer-sphere metal–phosphate complexes. At high levels of divalent metal ions, and when the metal ion concentration exceeds the DNA–phosphate concentration, the fraction of inner-sphere phosphate binding increases. In the presence of very high levels of Mg2+ (e.g., 3.1M), the inner-sphere ? outer-sphere equilibrium is shifted toward ~100% inner-sphere binding. A comparison of our DNA results and previous results obtained with tRNA indicates that tRNA and DNA have very similar divalent metal ion binding properties. A comparison of the present results with the predictions of polyelectrolyte theories is presented.  相似文献   

17.
To provide molecular-level insights into the spontaneous replication error and the mismatch discrimination mechanisms of human DNA polymerase β (polβ), we report four crystal structures of polβ complexed with dG•dTTP and dA•dCTP mismatches in the presence of Mg2+ or Mn2+. The Mg2+-bound ground-state structures show that the dA•dCTP-Mg2+ complex adopts an ‘intermediate’ protein conformation while the dG•dTTP-Mg2+ complex adopts an open protein conformation. The Mn2+-bound ‘pre-chemistry-state’ structures show that the dA•dCTP-Mn2+ complex is structurally very similar to the dA•dCTP-Mg2+ complex, whereas the dG•dTTP-Mn2+ complex undergoes a large-scale conformational change to adopt a Watson–Crick-like dG•dTTP base pair and a closed protein conformation. These structural differences, together with our molecular dynamics simulation studies, suggest that polβ increases replication fidelity via a two-stage mismatch discrimination mechanism, where one is in the ground state and the other in the closed conformation state. In the closed conformation state, polβ appears to allow only a Watson–Crick-like conformation for purine•pyrimidine base pairs, thereby discriminating the mismatched base pairs based on their ability to form the Watson–Crick-like conformation. Overall, the present studies provide new insights into the spontaneous replication error and the replication fidelity mechanisms of polβ.  相似文献   

18.
Abstract

The crystal structures of five double helical DNA fragments containing non-Watson-Crick complementary base pairs are reviewed. They comprise four fragments containing G·T base pairs: two deoxyoctamers d(GGGGCTCC) and d(GGGGTCCC) which crystallise as A type helices; a deoxydodecamer d(CGCGAATTTGCG) which crystallises in the B-DNA conformation; and the deoxyhexamer d(TGCGCG), which crystallises as a Z-DNA helix. In all four duplexes the G and T bases form wobble base pairs, with bases in the major tautomer forms and hydrogen bonds linking N1 of G with 02 of T and 06 of G with N3 of T. The X-ray analyses establish that the G·T wobble base pair can be accommodated in the A, B or Z double helix with minimal distortion of the global conformation. There are, however, changes in base stacking in the neighbourhood of the mismatched bases. The fifth structure, d(CGCGAATTAGCG), contains the purine purine mismatch G·A where G is in the anti and A in the syn conformation. The results represent the first direct structure determinations of base pair mismatches in DNA fragments and are discussed in relation to the fidelity of replication and mismatch recognition.  相似文献   

19.
RNA polymerase has been solubilized from sugar beet chromatin. With calf thmus or sugar beet DNA as template enzyme activity was linear with respect to protein concentration and required the presence of all four nucleoside triphospahates, added DNA and divalent metal ions. The enzyme exhibited a sharp Mn2+ optimum of 1·25 mM and a Mg2+ optimum at 10mM. The Mn2+/Mg2+ activity ratio (activity at optimum concentrations) was 2·0 with an optimum salt concentration of 50 mM. Based on data including inhibition with α-amanitin (0·025 μg/ml), the majority of the total activity appeared to be RNA polymerase I. Subsequent fractionation by DEAE-Sephadex column chromatography resulted in one peak of activity eluted with 0·18 M (NH4)2SO4.  相似文献   

20.
The effect of Mn2+, a known mutagen, on the fidelity of DNA synthesis in vitro by avian myeloblastosis DNA polymerase has been determined. Substitution of Mn2+ for Mg2+ leads to an enhanced incorporation of noncomplementary deoxynucleotides as well as complementary ribonucleotides with either poly (A) or poly (C) as templates. Since this polymerase lacks any detectable deoxyribonuclease activity, the in vitro mutagenic effect of Mn2+ in promoting errors in base-pairing does not result from any diminished proof-reading function.  相似文献   

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