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1.
2.
Naturally occurring abasic sites in DNA exist as an equilibrium mixture of the aldehyde, the hydrated aldehyde, and the hemiacetal forms (dominant). The influence of the configuration of the C1' hydroxyl group of the hemiacetal form on duplex structure and abasic site repair has been examined using novel carbocyclic analogues. Both the alpha- and beta-forms of this novel abasic site were introduced into oligomeric DNA using the standard DMT-phosphoramidite approach in an automated solid-phase synthesizer. Solution structures of the d(CGTACXCATGC).d(GCATGAGTACG) duplex (where X is the alpha- or beta-anomer of the carbocyclic abasic site analogue) were determined by NMR spectroscopy and restrained molecular dynamics simulations. The structures were only minimally perturbed by the presence of either anomer of the abasic site. All residues adopted an anti conformation, and Watson-Crick alignments were observed on all base pairs of the duplexes. At the lesion site, the abasic residues and their partner adenines showed increased dynamic behavior but adopted intrahelical positions in the final refined structures. Incision of duplexes having the alpha- or beta-anomer of the carbocyclic abasic site by human AP endonuclease showed that the enzyme recognizes both configurations of the lesion and nicks the DNA backbone with similar efficiency. Our results challenge the suggestion that Ape1 is stereoselective and imply a plasticity at the active site of the enzyme for accommodating either anomer of the lesion.  相似文献   

3.
Pseudorotationally locked sugar analogues based on bicyclo[3.1.0]-hexane templates were placed in DNA duplexes as abasic target sites in the M. HhaI recognition sequence. The binding affinity of the enzyme increases when the abasic site is constrained to the South conformation and decreases when it is constrained to the North conformation. A structural understanding of these differences is provided.  相似文献   

4.
Chen J  Dupradeau FY  Case DA  Turner CJ  Stubbe J 《Biochemistry》2007,46(11):3096-3107
A 4'-oxidized abasic site (X) has been synthesized in a defined duplex DNA sequence, 5'-d(CCAAAGXACCGGG)-3'/3'-d(GGTTTCATGGCCC)-5' (1). Its structure has been determined by two-dimensional NMR methods, molecular modeling, and molecular dynamics simulations. 1 is globally B-form with the base (A) opposite X intrahelical and well-stacked. Only the alpha anomer of X is observed, and the abasic site deoxyribose is largely intrahelical. These results are compared with a normal abasic site (Y) in the same sequence context (2). Y is composed of a 60:40 mixture of alpha and beta anomers (2alpha and 2beta). In both 2alpha and 2beta, the base (A) opposite Y is intrahelical and well-stacked and the abasic site deoxyribose is predominantly extrahelical, consistent with the reported structures of the normal abasic site in a similar sequence context [Hoehn, S. T., Turner, C. J., and Stubbe, J. (2001) Nucleic Acids Res. 29, 3413-3423]. Molecular dynamics simulations reveal that the normal abasic site appears to be conformationally more flexible than the 4'-oxidized abasic site. The importance of the structure and flexibility of the abasic site in the recognition by the DNA repair enzyme Ape1 is discussed.  相似文献   

5.
Three analogs of bradykinin and one of angiotensin II have been prepared in which the naturally occurring proline residues have been replaced by the bicyclic amino acid, 2,4-methanoproline (2,4-MePro). The relative binding affinities for these analogs were determined to be significantly reduced in the cases of the three bradykinin analogs; [2,4-MePro3]-BK retains 1.3%, [2,4-MePro7]-BK retains 0.3% and [2,4-MePro2]-BK retains 0.021% of the binding affinity of bradykinin. Results from other modification at positions three and seven indicate preference for the trans-amide bond preceding these residues implying that other factors, either steric or conformational, are responsible for the decreased affinity for the receptor seen with 2,4-MePro substitution. The retention of significant binding affinity (26%) in the case of [Ile5,2,4-MePro7]-angiotensin II gives direct evidence that the trans-conformation of the proline amide bond is the one recognized by the AII receptor. Only significant retention of activity can be interpreted unambiguously with the use of this proline analog because of its known conformational differences from Pro as well as its increased steric requirements at the receptor.  相似文献   

6.
Translesional DNA synthesis past abasic sites proceeds with the preferential incorporation of dAMP opposite the lesion and, depending on the sequence context, one or two base deletions. High-resolution NMR spectroscopy and molecular dynamics simulations were used to determine the three-dimensional structure of a DNA heteroduplex containing a synthetic abasic site (tetrahydrofuran) residue positioned in a sequence that promotes one base deletions. Analysis of NMR spectra indicates that the stem region of the duplex adopts a right-handed helical structure and the glycosidic torsion angle is in anti orientation for all residues. NOE interactions establish Watson-Crick alignments for all canonical base pairs of the duplex. Measurement of distance interactions at the lesion site shows the abasic residue excluded from the helix. Restrained molecular dynamics simulations generated three-dimensional models in excellent agreement with the spectroscopic data. These structures show a regular duplex region and a slight bend at the lesion site. The tetrahydrofuran residue extrudes from the helix and is highly flexible. The model reported here, in conjunction with a previous study performed on abasic sites, explains the structural bias of one-base deletion mutations.  相似文献   

7.
The conformational tendencies of C(alpha,alpha)-diethylglycine (Deg)-based peptides have been studied in solution using all atom molecular dynamics simulations. Specifically, the conformational effects of breaking the symmetry of the host Tfa-(Deg)(5)-OtBu (Tfa, trifluoroacetyl; OtBu, tert-butoxy) pentapeptide with punctual replacements at different sequence positions of one Deg residue by its corresponding guest chiral analogue, L-alpha-aminobutyric acid (L-Abu), have been examined by simulating the following peptides: Tfa-(Deg)(5)-OtBu, Tfa-(Deg)(2)-L-Abu-(Deg)(2)-OtBu, Tfa-(Deg)(3)-L-Abu-Deg-OtBu, and Tfa-(Deg)(4)-L-Abu-OtBu. Simulations show that only the Deg homopeptide is able to stabilize a 2.0(5) helix, even though a kinked arrangement with all the Deg residues adopting a fully-extended conformation was found to be stable when the L-Abu residue is introduced in the middle of the sequence. On the other hand, when the L-Abu residue is closer to the C-end of the sequence, the peptide chain prefers a partially folded 3(10)-helix. Additional simulations on Tfa-(Deg)(3)-L-Abu-(Deg)(3)-OtBu highlighted that, when the size of the Deg segments increases, their tendency to adopt a 2.0(5) helix predominates over the preferred folded conformation of L-Abu. The overall picture extracted after more than 300 ns of molecular dynamics simulation is that breaking the alpha-carbon symmetry of achiral C(alpha)-tetrasubstituted amino acids is a promising strategy to build up polypeptides with modulated conformational tendencies.  相似文献   

8.
Mutagenesis at abasic sites was investigated in E.coli and simian kidney (COS) cells using a duplex shuttle vector containing synthetic analogs of deoxyribose on the phosphodiester backbone. Lesions were positioned on opposite strands of the vector. When the tetrahydrofuranyl analog was used as the abasic site, AT or TA pairs (65-80%) were introduced at the site of the bistrand lesion. Mutagenesis occurred in the absence of SOS induction. Single base deletions (> 80%) dominated the mutational spectra for propanyl and ethanyl analogs of abasic sites lacking a ring structure. For all abasic site analogs, a small proportion of G/C and C/G pairs (6-10%) were observed. dAMP was incorporated predominantly opposite tetrahydrofuranyl sites positioned in the single strand region of a gapped duplex vector. We conclude from these studies that abasic sites positioned in a bistrand configuration are highly mutagenic in E.coli and COS cells. Repair DNA synthesis may be involved in this process.  相似文献   

9.
The synthetic dodecanucleotide d(CGCAAATTGGCG) has been analysed by single-crystal X-ray diffraction techniques and the structure refined to R = 0.16 and 2.25 A resolution, with the location of 94 solvent molecules. The sequence crystallizes as a full turn of a B-DNA helix with ten Watson-Crick base-pairs and two adenine-guanine mispairs. The analysis clearly shows that the mismatches are of the form A(anti).G(syn). Thermal denaturation studies indicate that the stability of the duplex is strongly pH dependent, with a maximum at pH 5.0, suggesting that the base-pair is stabilized by protonation. Three different arrangements have been observed for base-pairs between guanine and adenine and it is likely that A.G mismatch conformation is strongly influenced by dipole-dipole interactions with adjacent base-pairs.  相似文献   

10.
F Seela  Y Chen 《Nucleic acids research》1995,23(13):2499-2505
The fluorescent nucleoside 2'-deoxyisoinosine (2, isoId) has been incorporated into oligonucleotides. For this purpose the phosphonate 3a and the phosphoramidite 3b, as well as the polymer-linked 3d, have been synthesized and oligonucleotides were prepared by P(III) solid-phase chemistry. One or two isoId-residues were introduced into the oligomer d(T12), replacing dT either in the middle or at the 3'- and 5'-ends. The isoId-containing oligomers were hybridized with a modified d(A)12 containing the conventional nucleosides (dA, dT, dG and dC) opposite to isoId. The replacement of one dT by isoId in the centre of the duplex reduced the Tm value by approximately 15 degrees C and a decrease of approximately 25 degrees C was found when two isoId residues were incorporated. Thermodynamic data were determined from the melting curves. The destabilization was almost independent of the four naturally occurring nucleosides located opposite to isoId. The isoId (2) seems to be stacked in the duplex when dT-dA base pairs are the nearest neighbours; an internal loop is formed in the case of oligomers containing two consecutive isold residues.  相似文献   

11.
The flexibility and curvature of duplex DNAs containing mismatched sites have been monitored as a function of temperature. The diffusion coefficients are dependent on the flexibility and the curvature of the DNA, and these have been determined by NMR-based methods. The diffusion coefficients, D, depend on a Boltzmann term and the viscosity of the solvent, eta, which is also temperature dependent. To analyze the temperature dependence of the diffusion results, the shape function, S(f) = etaD/T, is used. The shape functions do not have the viscosity and temperature dependence of the diffusion coefficients. The presence of mismatched sites significantly enhances the shape function of duplex DNA at all temperatures examined. The observed increases in the shape functions are attributed to the mismatched sites acting as localized flexible joints. The results on the temperature dependence of the shape functions, the optical absorbance, and the proton chemical shifts indicate that local melting at, and adjacent to, mismatched site occurs at a lower temperature than the overall melting of the duplexes. The localized melting gives rise to a considerable increase in the shape function. The contribution of the curvature of the mismatched sites to the enhanced diffusion has been examined. A DNA with mismatches that are in phase with respect to the helical repeat and a DNA which has the mismatches out of phase with respect to the helical repeat have been examined. The results indicate that mismatched sites have modest curvature.  相似文献   

12.
We investigated the effects of formaldehyde fixation on the secondary structure of isolated proteins (bovine serum albumin, ribonuclease A, and hemoglobin) using high-sensitivity differential scanning calorimetry and Fourier transform infrared spectroscopy. Whereas thermograms obtained by scanning calorimetry on unfixed purified proteins demonstrated denaturation transitions in the 70-90 degrees C temperature range, the thermograms showed no denaturation transitions in this temperature range when the proteins had been placed in formaldehyde solutions. Thus, fixation destroyed the denaturation transition of bovine serum albumin, ribonuclease A, and hemoglobin. Infrared spectra obtained on the unfixed and fixed proteins were essentially identical. This demonstrates that the "fixed" proteins retain the secondary structure present before fixation. We therefore conclude that the cross-linking of proteins that occurs in the process of formaldehyde fixation "locks in" the secondary structure of these protein molecules.  相似文献   

13.
The three-dimensional solution structure of two DNA decamers of sequence d(CCACXGGAAC)-(GTTCCGGTGG) with a modified nucleotide containing a cholesterol derivative (X) in its C1 '(chol)alpha or C1 '(chol)beta diastereoisomer form has been determined by using NMR and restrained molecular dynamics. This DNA derivative is recognized with high efficiency by the UvrB protein, which is part of the bacterial nucleotide excision repair, and the alpha anomer is repaired more efficiently than the beta one. The structures of the two decamers have been determined from accurate distance constraints obtained from a complete relaxation matrix analysis of the NOE intensities and torsion angle constraints derived from J-coupling constants. The structures have been refined with molecular dynamics methods, including explicit solvent and applying the particle mesh Ewald method to properly evaluate the long range electrostatic interactions. These calculations converge to well defined structures whose conformation is intermediate between the A- and B-DNA families as judged by the root mean square deviation but with sugar puckerings and groove shapes corresponding to a distorted B-conformation. Both duplex adducts exhibit intercalation of the cholesterol group from the major groove of the helix and displacement of the guanine base opposite the modified nucleotide. Based on these structures and molecular dynamics calculations, we propose a tentative model for the recognition of damaged DNA substrates by the UvrB protein.  相似文献   

14.
M Bjors  L Luna  B Johnsen  E Hoff  T Haug  T Rognes    E Seeberg 《The EMBO journal》1997,16(20):6314-6322
The guanine modification 7,8-dihydro-8-oxoguanine (8-oxoG) is a potent premutagenic lesion formed spontaneously at high frequencies in the genomes of aerobic organisms. We have characterized a human DNA repair glycosylase for 8-oxoG removal, hOGH1 (human yeast OGG1 homologue), by molecular cloning and functional analysis. Expression of the human cDNA in a repair deficient mutator strain of Escherichia coli (fpg mutY) suppressed the spontaneous mutation frequency to almost normal levels. The hOGH1 enzyme was localized to the nucleus in cells transfected by constructs of hOGH1 fused to green fluorescent protein. Enzyme purification yielded a protein of 38 kDa removing both formamidopyrimidines and 8-oxoG from DNA. The enzymatic activities of hOGH1 was analysed on DNA containing single residues of 8-oxoG or abasic sites opposite each of the four normal bases in DNA. Excision of 8-oxoG opposite C was the most efficient and was followed by strand cleavage via beta-elimination. However, significant removal of 8-oxoG from mispairs (8-oxoG: T >G >A) was also demonstrated, but essentially without an associated strand cleavage reaction. Assays with abasic site DNA showed that strand cleavage was indeed dependent on the presence of C in the opposite strand, irrespective of the prior removal of an 8-oxoG residue. It thus appears that strand incisions are made only if repair completion results in correct base insertion, whereas excision from mispairs preserves strand continuity and hence allows for error-free correction by a postreplicational repair mechanism.  相似文献   

15.
A 13 mers abasic oligonucleotide was synthetized. It was therefore possible to compare thermal stability and reactivity of duplex oligonucleotides either with an apurinic/apyrimidinic site or without any lesion. An important decrease in the melting temperature appeared for duplexes with an abasic site. The chemical reaction of these modified oligonucleotides with the intercalating agent 9-aminoellipticine was studied by gel electrophoresis and by fluorescence. The formation of a Schiff base between 9-aminoellipticine and abasic sites was rapid and complete with duplexes at 11 degrees C. Schiff base related fluorescence and beta-elimination cleavage were more important with the apyrimidinic sites than with the apurinic ones. When compared to previous results obtained with the model d(TprpT) some unexpected behaviours appeared with longer and duplex oligonucleotides. For instance only partial beta-elimination cleavage was observed. It is likely that stacking parameters in the double helix play a great role in the studied reaction.  相似文献   

16.
A comparative study of interaction between chicken egg white lysozyme (Lyz) with two hexavalent chromate ions; chromate and dichromate; which are prevalently known for their toxicity, was investigated using different spectroscopic techniques along with a molecular docking study. Both steady-state and time-resolved studies revealed that the addition of chromate/dichromate is responsible for strong quenching of intrinsic fluorescence in Lyz and the quenching is caused by both static and dynamic quenching mechanisms. Different binding and thermodynamic parameters were also calculated at different temperatures from the intrinsic fluorescence of Lyz. The conformational change in Lyz and thermodynamic parameters obtained during the course of interaction with chromate/dichromate were well-supported by the molecular docking results.  相似文献   

17.
Metal-ion and sequence dependent changes in the stacking interactions of bases surrounding abasic (AB) sites in 10 different DNA duplexes were examined by incorporating the fluorescent nucleotide probe 2-aminopurine (2-AP), opposite to the site (AB-APopp) or adjacent to the site (AB-APadj) on either strand. A detailed study of the fluorescence emission and excitation spectra of these AB duplexes and their corresponding parent duplexes indicates that AB-APoppis significantly less stacked than 2-AP in the corresponding normal duplex. In general, AB-APadjon the AB strand is stacked, but AB-APadjon the opposite strand shows destabilized stacking interactions. The results also indicate that divalent cation binding to the AB duplexes contributes to destabilizaton of the base stacking interactions of AB-APopp, but has little or no effect on the stacking interactions of AB-APadj. Consistent with these results, the fluorescence of AB-APoppis 18-30-fold more sensitive to an externally added quenching agent than the parent normal duplex. When uracil DNA glycosylase binds to AB-APoppin the presence of 2.5 mM MgCl2, a 3-fold decrease in fluorescence is observed ( K d = 400 +/- 90 nM) indicating that the unstacked 2-APoppbecomes more stacked upon binding. On the basis of these fluorescence studies a model for the local base stacking interactions at these AB sites is proposed.  相似文献   

18.
-Melanotropin and ACTH, POMC peptides, initiate biological activity by interaction with the classical pigment cell (-MSH receptor, MC1R) and adrenal gland (ACTH receptor, MC2R) melanocortin receptors, respectively. The recently discovered MC3R, MC4R and MC5R receptors provide new targets and new biological functions for POMC peptides. We have developed conformationally constrained -melanotropin peptides that interact with all of these receptors as agonists and antagonists and are examining new approaches to obtain highly selective ligands for each of these melanocortin receptors. Previously, we had converted somatostatin-derived peptides into potent and highly selective analogues that act as antagonists at the opioid receptors. Using the reverse turn template that came out of these studies, we have designed, de novo, agonist and antagonist peptide analogues that interact with melanocortin receptors.  相似文献   

19.
Summary α-Melanotropin and ACTH, POMC peptides, initiate biological activity by interaction with the classical pigment cell (α-MSH receptor, MC1R) and adrenal gland (ACTH receptor, MC2R) melanocortin receptors, respectively. The recently discovered MC3R, MC4R and MC5R receptors provide new targets and new biological functions for POMC peptides. We have developed conformationally constrained α-melanotropin peptides that interact with all of these receptors as agonists and antagonists and are examining new approaches to obtain highly selective ligands for each of these melanocortin receptors. Previously, we had converted somatostatin-derived peptides into potent and highly selective analogues that act as antagonists at the μ opioid receptors. Using the reverse turn template that came out of these studies, we have designed, de novo, agonist and antagonist peptide analogues that interact with melanocortin receptors.  相似文献   

20.
New analogues of deltorphin I (DT I, Tyr‐d ‐Ala‐Phe‐Asp‐Val‐Val‐Gly‐NH2), with the d ‐Ala residue in position 2 replaced by α‐methyl‐β‐azido(amino, 1‐pyrrolidinyl, 1‐piperidinyl or 4‐morpholinyl)alanine, were synthesized by a combination of solid‐phase and solution methods. All ten new analogues were tested for receptor affinity and selectivity to μ‐ and δ‐opioid receptors. The affinity of analogues containing (R) or (S)‐α‐methyl‐β‐azidoalanine in position 2 to δ‐receptors strongly depended on the chirality of the α,α‐disubstituted residue. Peptide II , containing (S)‐α‐methyl‐β‐azidoalanine in position 2, displayed excellent δ‐receptor selectivity with its δ‐receptor affinity being only three times lower than that of DT I. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.  相似文献   

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