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1.
《Nucleosides, nucleotides & nucleic acids》2013,32(5-8):653-658
Abstract Nucleoside analogs such as 1-β-D-arabinofuranosyl cytidine (AraC) and 2′, 2′-difluoro deoxycytidine (dFdC) are important components of the anticancer chemotherapeutic arsenal and are among the most effective anticancer drugs currently available. Although both AraCTP and dFdCTP impede DNA replication through pausing of DNA polymerases, both nucleoside analogs are ultimately incorporated into replicated DNA and interfere in DNA-mediated processes. Our laboratories are investigating the structural basis for the poisoning of topoisomerase I (top1) due to antipyrimidine incorporation into duplex DNA. We recently reported that both AraC and dFdC induce formation of top1 cleavage complexes, and poisoning of top1 contributes to the anticancer activities of both these drugs. Recent NMR and thermodynamic studies from our laboratories provide insight into the mechanism by which AraC and dFdC poison top 1. NMR studies from our laboratories have revealed that the arabinosyl sugar of AraC adopted a C2′-endo conformation. Although this is a B-type sugar pucker characteristic of duplex DNA, the conformation is rigid, and this lack of flexibility probably contributes to inhibition of the religation step of the top 1 reaction. In contrast to AraC, NMR studies revealed dFdC adopted a C3′ endo sugar pucker characteristic of RNA, rather than DNA duplexes. dFdC substitution enhanced formation of top1 cleavage complexes, but did not inhibit religation. The enhancement of top1 cleavage complexes most likely results from a combination of conformational and electrostatic effects. The structural effects of dFdC and AraC are being further investigated in duplex DNA with well-defined top1 cleavage sites to analyze more specifically how these structural perturbations lead to enzyme poisoning. 相似文献
2.
Antimetabolite incorporation into DNA: structural and thermodynamic basis for anticancer activity 总被引:1,自引:0,他引:1
Gmeiner WH 《Biopolymers》2002,65(3):180-189
Antimetabolites are a class of effective anticancer drugs that structurally resemble naturally occurring biochemicals and interfere in essential biochemical processes. In this review, the recent literature describing investigations of the structural and thermodynamic basis for the anticancer activity of three antipyrimidines [1-beta-D-arabinofuranosyl cytidine (AraC). 2',2'-difluoro deoxycytidine (dFdC), and 5-fluoro-2'-deoxyuridine (FdUrd)] is summarized. Our laboratory, and others, have shown that misincorporation of any of these three antipyrimidines into DNA perturbs the structure and decreases the stability of duplex DNA. These data are useful for rationalizing the effects of antipyrimidine misincorporation on the activities of proteins required for DNA replication and repair such as DNA topoisomerase 1 and DNA polymerases. The studies completed to date and summarized in this review demonstrate the utility of investigations into the structure-function relationships between antipyrimidine-substituted DNA complexed with DNA-modifying proteins for the purpose of understanding the basis for effective antipyrimidine cancer chemotherapy and the future design of novel anticancer drugs. 相似文献
3.
Miguel Garcia-Diaz Michael S. Murray Thomas A. Kunkel Kai-ming Chou 《The Journal of biological chemistry》2010,285(22):16874-16879
The anticancer activity of cytarabine (AraC) and gemcitabine (dFdC) is thought to result from chain termination after incorporation into DNA. To investigate their incorporation into DNA at atomic level resolution, we present crystal structures of human DNA polymerase λ (Pol λ) bound to gapped DNA and containing either AraC or dFdC paired opposite template dG. These structures reveal that AraC and dFdC can bind within the nascent base pair binding pocket of Pol λ. Although the conformation of the ribose of AraCTP is similar to that of normal dCTP, the conformation of dFdCTP is significantly different. Consistent with these structures, Pol λ efficiently incorporates AraCTP but not dFdCTP. The data are consistent with the possibility that Pol λ could modulate the cytotoxic effect of AraC. 相似文献
4.
Thiviyanathan V Guliaev AB Leontis NB Gorenstein DG 《Journal of molecular biology》2000,300(5):1143-1154
Bulges are common structural motifs in RNA secondary structure and are thought to play important roles in RNA-protein and RNA-drug interactions. Adenosine bases are the most commonly occurring unpaired base in double helical RNA secondary structures. The solution conformation and dynamics of a 25-nucleotide RNA duplex containing an unpaired adenosine, r(GGCAGAGUGCCGC): r(GCGGCACCUGCC) have been studied by NMR spectroscopy and MORASS iterative relaxation matrix structural refinement. The results show that the bulged adenosine residue stacks into the RNA duplex with little perturbation around the bulged region. Most of the bases in the RNA duplex adopt C(3)'-endo conformation, exhibiting the N-type sugar pucker as found in the A form helices. The sugars of the bulged residue and the 5' flanking residue to it are found to exhibit C(2)'-endo conformation. None of the residues are in syn conformation. 相似文献
5.
Conformational microheterogeneity in a DNA double helix: structure of restriction endonuclease Bam H1 recognition site 总被引:1,自引:0,他引:1
M H Sarma M M Dhingra G Gupta R H Sarma 《Biochemical and biophysical research communications》1985,131(1):269-276
Structural studies using 500 MHz 1H NMR spectroscopy on Bam H1 recognition site d(GGATCC)2 in solution at 19 degrees is reported. The resonances from the sugar ring and base protons have been assigned from the 2D-COSY and NOESY spectra. Analyses of the NOESY cross-peaks between the base protons H8/H6 and sugar protons H2'/H2", H3' reveal that the nucleotide units G2, A3 and C6 adopt (C3'-endo, chi = 200 degrees-220 degrees) conformation while G1, T4 and C5 exhibit (C2'-endo, chi = 240 degrees-260 degrees) conformation. NMR data clearly suggest that the two strands of d(GGATCC)2 are conformationally equivalent and there is a structural two-fold between the two A-T pairs. The above information and the NOESY data are used to generate a structural model of d(GGATCC)2. The important features are: (i) G1-G2 stack, the site of cleavage, shows an alternation in sugar pucker i.e. C2'-endo, C3'-endo as in a B-A junction, (ii) G2-A3 stack adopts a mini A-DNA, both the sugars being C3'-endo, (iii) A3-T4 stack, the site of two-fold, displays an A-B junction with alternation in sugar pucker as C3'-endo, C2'-endo, (iv) T4-C5 stack adopts a mini B-DNA both the sugars being C2'-endo and (v) C5-C6 stack exhibits a B-A junction with C2'-endo, C3'-endo sugar puckers. Thus, our studies demonstrate that conformational microheterogeneity with a structural two fold, is present in the Bam H1 recognition site. 相似文献
6.
Chrencik JE Burgin AB Pommier Y Stewart L Redinbo MR 《The Journal of biological chemistry》2003,278(14):12461-12466
1-beta-d-Arabinofuranosylcytosine (Ara-C) is a potent antineoplastic drug used in the treatment of acute leukemia. Previous biochemical studies indicated the incorporation of Ara-C into DNA reduced the catalytic activity of human topoisomerase I by decreasing the rate of single DNA strand religation by the enzyme by 2-3-fold. We present the 3.1 A crystal structure of human topoisomerase I in covalent complex with an oligonucleotide containing Ara-C at the +1 position of the non-scissile DNA strand. The structure reveals that a hydrogen bond formed between the 2'-hydroxyl of Ara-C and the O4' of the adjacent -1 base 5' to the damage site stabilizes a C3'-endo pucker in the Ara-C arabinose ring. The structural distortions at the site of damage are translated across the DNA double helix to the active site of human topoisomerase I. The free sulfhydryl at the 5'-end of the nicked DNA strand in this trapped covalent complex is shifted out of alignment with the 3'-phosphotyrosine linkage at the catalytic tyrosine 723 residue, producing a geometry not optimal for religation. The subtle structural changes caused by the presence of Ara-C in the DNA duplex may contribute to the cytotoxicity of this leukemia drug by prolonging the lifetime of the covalent human topoisomerase I-DNA complex. 相似文献
7.
W H Gmeiner K E Rao B Rayner J J Vasseur F Morvan J L Imbach J W Lown 《Biochemistry》1990,29(45):10329-10341
The novel hybrid duplex alpha-5'-d[TACACA]-3'.beta-5'-r[AUGUGU]-3' was analyzed extensively by 1D and 2D NMR methods. Two forms of the duplex exist in about an 80:20 ratio. Analysis of the exchangeable imino protons of the major component revealed that three AU and one AT base pair are present in addition to two GC base pairs, confirming that the duplex anneals in parallel orientation. The presence of the AT base pair, which can only be accounted for by a parallel duplex, was confirmed by a selective INEPT experiment, which correlated the thymidine imino proton to its C5 carbon. The lesser antiparallel form could be detected by exchangeable and nonexchangeable proton resonances in both strands. An exchange peak was observed in the NOESY spectrum for the thymidine methyl group resonance in both the predominant and lesser conformations, indicating the lifetime of the individual structures was on the millisecond time scale. The nonexchangeable protons of the predominant duplex were assigned by standard methods. The sugar pucker of the ribonucleosides was determined to be of the "S" type by a pseudorotation analysis according to Altona, with the J-couplings measured from the multiplet components of the phase-sensitive COSY experiment. The NOE pattern observed for the alpha-deoxynucleosides also suggested an S-type sugar pucker. The adoption of an S-type sugar pucker for both strands indicates that, in contrast to RNA.DNA duplexes formed exclusively from beta-nucleotides, the alpha-DNA.beta-RNA duplex may form a B-type helix. The 31P resonances of the alpha and beta strands have very different chemical shifts in the hybrid duplex and the difference persists above the helix melting temperature, indicating an intrinsic difference in 31P chemical shift for nucleotides differing only in the configuration about the glycosidic bond. 相似文献
8.
Pommier Y Kohlhagen G Laco GS Kroth H Sayer JM Jerina DM 《The Journal of biological chemistry》2002,277(16):13666-13672
Topoisomerase I (top1) relieves supercoiling in DNA by forming transient covalent cleavage complexes. These cleavage complexes can accumulate in the presence of damaged DNA or anticancer drugs that either intercalate or lie in the minor groove. Recently we reported that covalent diol epoxide (DE) adducts of benzo[a]pyrene (BaP) at the exocyclic amino group of G(+1) block cleavage at a preferred cleavage site ( approximately CTT-G(+1)G(+2)A approximately ) and cause accumulation of cleavage products at remote sites. In the present study, we have found that the 10S G(+2) adduct of BaP DE, which lies toward the scissile bond in the minor groove, blocks normal cleavage, whereas the 10R isomer, which orients away from this bond, allows normal cleavage but blocks religation. In contrast to BaP, the pair of benzo[c] phenanthrene (BcPh) DE adducts at G(+2), which intercalate from the minor groove either between G(+1)/G(+2) or between G(+2)/A, allow normal cleavage but block religation. Both intercalated BcPh DE adducts at G(+1) suppress normal cleavage, as do both groove bound BaP DE adducts at this position. These studies demonstrate that these DE adducts provide a novel set of tools to study DNA topoisomerases and emphasize the importance of contacts between the minor groove and top1's catalytic site. 相似文献
9.
Topoisomerase I (Top1) activities are sensitive to various endogenous base modifications, and anticancer drugs including the natural alkaloid camptothecin. Here, we show that triple helix-forming oligonucleotides (TFOs) can enhance Top1-mediated DNA cleavage by affecting either or both the nicking and the closing activities of Top1 depending on the position and the orientation of the triplex DNA structure relative to the Top1 site. TFO binding 1 bp downstream from the Top1 site enhances cleavage by inhibiting religation and to a lesser extent DNA nicking. In contrast, TFO binding 4 bp downstream from the Top1 site enhances DNA nicking especially when the 3′ end of the TFO is proximal to the Top1 site. However, when the orientation of the triplex is inverted, with its 5′ terminus 4 bp downstream from the Top1 site, religation is also inhibited. These position- and orientation-dependent effects of triplex structures on the Top1-mediated DNA cleavage and religation are discussed in the context of molecular modeling and effects of TFO on DNA twist and mobility at the duplex/triplex junction. 相似文献
10.
Studies of the topoisomerase II-mediated cleavage and religation reactions by use of a suicidal double-stranded DNA substrate. 总被引:6,自引:0,他引:6
A H Anderson B S S?rensen K Christiansen J Q Svejstrup K Lund O Westergaard 《The Journal of biological chemistry》1991,266(14):9203-9210
The cleavage and religation reactions of eukaryotic topoisomerase II were studied by use of a 5'-recessed DNA substrate containing a strong recognition sequence for the enzyme. Cleavage of the DNA substrate was suicidal, that is the enzyme was unable to religate the cleaved DNA due to a release of DNA 5' to the cleavage position. With this substrate cleavage products accumulated with time in the absence of protein-denaturing agents, and the cleavage reaction was not reversible with salt. The suicide cleavage complexes contained a kinetically competent topoisomerase II enzyme as determined by the enzyme's ability to perform intermolecular ligation of the cleaved DNA to a free 3'-hydroxyl end on another DNA strand. The efficiency of the religation reaction depended on the ability of the religation substrate to base pair to the DNA in the cleaved enzyme-DNA complex. Higher levels of religation were obtained with dinucleotides than with long DNA substrates. Mononucleotides also were efficiently religated, indicating an ability of the enzyme to mediate religation without making contacts to a long stretch of nucleotides 5' to the cleavage position. 相似文献
11.
12.
Barthwal R Monica Awasthi P Srivastava N Sharma U Kaur M Govil G 《Journal of biomolecular structure & dynamics》2003,21(3):407-423
Solution conformation of self-complementary DNA duplex d-CGATCG, containing 5' d-CpG 3' site for intercalation of anticancer drug, daunomycin and adriamycin, has been investigated by nuclear magnetic resonance (NMR) spectroscopy. Complete resonance assignments of all the protons (except some H5'/H5" protons) have been obtained following standard procedures based on double quantum filtered correlation spectroscopy (dQF COSY) and two-dimensional nuclear Overhauser effect (NOE) spectra. Analysis of sums of coupling constants in one-dimensional NMR spectra, cross peak patterns in dQF COSY spectra and inter proton distances shows that the DNA sequence assumes a conformation close to the B-DNA family. The deoxyribose sugar conformation is in dynamic equilibrium with predominantly S-type conformer and a minor N-type conformer with N<-->S equilibrium varying with temperature. At 325 K, the mole fraction of the N-conformer increases for some of the residues by approximately 9%. Using a total of 10 spin-spin coupling constants and 112 NOE intensities, structural refinement has been carried out using Restrained Molecular Dynamics (rMD) with different starting structures, potential functions and rMD protocols. It is observed that pseudorotation phase angle of deoxyribose sugar for A3 and T4 residues is approximately 180 degrees and approximately 120 degrees, respectively while all other residues are close to C2'endo-conformation. A large propeller twist (approximately -18 degrees) and smallest twist angle (approximately 31 degrees) at A3pT4 step, in the middle of the sequence, a wider (12 A) and shallower (3.0 A) major groove with glycosidic bond rotation as high anti at both the ends of hexanucleotide are observed. The structure shows base-sequence dependent variations and hence strong local structural heterogeneity, which may have implications in ligand binding. 相似文献
13.
An oligodeoxyribonucleotide N3'--> P5' phosphoramidate duplex forms an A-type helix in solution.
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D Ding S M Grayaznov D H Lloyd S Chandrasekaran S Yao L Ratmeyer Y Pan W D Wilson 《Nucleic acids research》1996,24(2):354-360
The solution conformations of the dinucleotide d(TT) and the modified duplex d(CGCGAATTCGCG)2 with N3'--> P5' phosphoramidate internucleoside linkages have been studied using circular dichroism (CD) and NMR spectroscopy. The CD spectra indicate that the duplex conformation is similar to that of isosequential phosphodiester RNA, a A-type helix, and is different from that of DNA, a B-type helix, NMR studies of model dimers d(TpT) and N3'--> P5' phosphoramidate d(TnpT) show that the sugar ring conformation changes from predominantly C2'-endo to C3'-endo when the 3'-phosphoester is replaced by a phosphoramidate group. Two-dimensional NMR (NOESY, DQF-COSY and TOCSY spectra) studies of the duplex provide additional details about the A-type duplex conformation of the oligonucleotide phosphoramidate and confirm that all furanose rings of 3'-aminonucleotides adopt predominantly N-type sugar puckering. 相似文献
14.
Determination of the DNA sugar pucker using 13C NMR spectroscopy 总被引:1,自引:0,他引:1
Solid-state 13C NMR spectroscopy of a series of crystalline nucleosides and nucleotides allows direct measurement of the effect of the deoxyribose ring conformation on the carbon chemical shift. It is found that 3'-endo conformers have 3' and 5' chemical shifts significantly (5-10 ppm) upfield of comparable 3'-exo and 2'-endo conformers. The latter two conformers may be distinguished by smaller but still significant differences in the carbon chemical shifts at the C-2' and C-4' positions. High-resolution solid-state NMR of three modifications of fibrous calf thymus DNA shows that these trends are maintained in high-molecular-weight DNA and confirms that the major ring pucker in A-DNA is 3'-endo, while both B-DNA and C-DNA are largely 2'-endo. The data show that 13C NMR spectroscopy is a straightforward and useful probe of DNA ring pucker in both solution and the solid state. 相似文献
15.
Keehwan Kwon Yu Lin Jiang Fenhong Song James T Stivers 《The Journal of biological chemistry》2002,277(1):353-358
The site-specific DNA cleavage and religation activities of the vaccinia virus type IB topoisomerase at (C/T)CCTT(+1)X(-1) sites in duplex DNA have allowed detailed investigations of the chemical and conformational steps on the reaction pathway of this enzyme (see accompanying article (Kwon, K., and Stivers, J. T. (2002) J. Biol. Chem. 277, 345-352)). To extend these studies to the DNA substrate, we have performed 19F NMR experiments using substrates in which the +1 T has been replaced with the NMR-sensitive thymidine base analogue 5-fluoro-2'-deoxyuridine (5-F-dUrd). Substitution of 5-F-dUrd has little effect on the binding affinity of topoisomerase I for DNA, results in small changes in the cleavage and religation rate constants, and produces a net 3-fold decrease in the cleavage equilibrium constant as compared with the CCCTT consensus DNA. One-dimensional 19F NMR experiments show that the +1 5-F-dUrd is in a dynamic equilibrium between a stacked and unstacked state in both the noncovalent complex and the covalent phosphotyrosine complex. These NMR observations are supported by the selective sensitivity of the +1 T and +1 5-F-dUrd to KMnO4 oxidation. A role for localized DNA distortion in the topoisomerase I mechanism is suggested. 相似文献
16.
Beauséjour CM Gagnon J Primeau M Momparler RL 《Biochemical and biophysical research communications》2002,293(5):1478-1484
Deoxycytidine nucleoside analogs must be first phosphorylated to become active anticancer drugs. The rate-limiting enzyme in this pathway is deoxycytidine kinase (dCK). Cells deficient in this enzyme are resistant to these analogs. To evaluate the potential of dCK to be used as suicide gene for deoxycytidine nucleoside analogs, we transduced both human A-549 lung carcinoma and murine NIH3T3 fibroblast cell lines with this gene. The dCK-transduced cells showed an increase in cytotoxicity to the analogs, cytosine arabinoside (ARA-C), and 5-aza-2'-deoxycytidine (5-AZA-CdR). Unexpectedly, the related analog, 2',2'-difluorodeoxycytidine (dFdC), was less cytotoxic to the dCK-transduced cells than the wild-type cells. For the A-549-dCK cells, the phosphorylation of dFdC by dCK was much greater than control cells. In accord with the elevated enzyme activity, we observed a 6-fold increased dFdC incorporation into DNA and a more pronounced inhibition of DNA synthesis in the A-549-dCK cells. In an attempt to clarify the mechanism of dFdC, we investigated its action on A549 and 3T3 cells transduced with both cytidine deaminase (CD) and dCK. We reported previously that overexpression of CD confers drug resistance to deoxycytidine analogs. In this study, when the CD-transduced cells were also transduced with dCK they became relatively more sensitive to dFdC. In addition, we observed that dFdU, the deaminated form of dFdC, was cytotoxic to the A-549-dCK cells, but not the wild-type cells. Our working hypothesis to explain these results is that the mitochondrial thymidine kinase (TK2), an enzyme reported to phosphorylate dFdC, acts as an important modulator of dFdC-induced cell toxicity. These findings may further clarify the action of dFdC and the mechanism by which it induces cell death. 相似文献
17.
The site-specific cleavage of synthetic Holliday junction analogs and related branched DNA structures by bacteriophage T7 endonuclease I 总被引:18,自引:0,他引:18
Various branched DNA structures were created from synthetic, partly complementary oligonucleotides combined under annealing conditions. Appropriate mixtures of oligonucleotides generated three specific branched duplex DNA molecules: (i) a Holliday junction analog having a fixed (immobile) crossover bounded by four duplex DNA branches, (ii) a similar Holliday junction analog which is capable of limited branch migration and, (iii) a Y-junction, with three duplex branches and fixed branch point. Each of these novel structures was specifically cleaved by bacteriophage T7 gene 3 product, endonuclease I. The cleavage reaction "resolved" the two Holliday structure analogs into pairs of duplex DNA products half the size of the original molecules. The point of cleavage in the fixed-junction molecules was predominantly one nucleotide removed to the 5' side of the expected crossover position. Multiple cleavage positions were mapped on the Holliday junction with the mobile, or variable, branch point, to sites consistent with the unrestricted movement of the phosphodiester crossover within the region of limited dyad symmetry which characterizes this molecule. Based on the cleavage pattern observed with this latter substrate, the enzyme displayed a modest degree of sequence specificity, preferring a pyrimidine on the 3' side of the cleavage site. Branched molecules that were partial duplexes (lower order complexes which possessed single-stranded as well as duplex DNA branches) were also substrates for the enzyme. In these molecules, the cleaved phosphodiester bonds were in duplex regions only and predominantly one nucleotide to the 5' side of the branch point. The phosphodiester positions 5' of the branch point in single-stranded arms were not cleaved. Under identical reaction conditions, individually treated oligonucleotides were completely refractory. Thus, cleavage by T7 endonuclease I displays great structural specificity with an efficiency that can vary slightly according to the DNA sequence. 相似文献
18.
We report on two-dimensional proton NMR studies of echinomycin complexes with the self-complementary d(A1-C2-G3-T4) and d(T1-C2-G3-A4) duplexes in aqueous solution. The exchangeable and nonexchangeable antibiotic and nucleic acid protons in the 1 echinomycin per tetranucleotide duplex complexes have been assigned from analyses of scalar coupling and distance connectivities in two-dimensional data sets recorded in H2O and D2O solution. An analysis of the intermolecular NOE patterns for both complexes combined with large upfield imino proton and large downfield phosphorus complexation chemical shift changes demonstrates that the two quinoxaline chromophores of echinomycin bisintercalate into the minor groove surrounding the dC-dG step of each tetranucleotide duplex. Further, the quinoxaline rings selectively stack between A1 and C2 bases in the d(ACGT) complex and between T1 and C2 bases in the d(TCGA) complex. The intermolecular NOE patterns and the base and sugar proton chemical shifts for residues C2 and G3 are virtually identical for the d(ACGT) and d(TCGA) complexes. A change in sugar pucker from the C2'-endo range to the C3'-endo range is detected at C2 on formation of the d(ACGT) and d(TCGA) complexes. In addition, the sugar ring protons of C2 exhibit upfield shifts and a large 1 ppm separation between the H2' and H2" protons for both complexes. The L-Ala amide protons undergo large downfield complexation shifts consistent with their participation in intermolecular hydrogen bonds for both tetranucleotide complexes.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
19.
3'-O-Anthraniloyladenosine, an analogue of the 3'- terminal aminoacyladenosine residue in aminoacyl-tRNAs, was prepared by chemical synthesis, and its crystal structure was determined. The sugar pucker of 3'-O-anthraniloyladenosine is 2'-endo resulting in a 3'-axial position of the anthraniloyl residue. The nucleoside is insynconformation, which is stabilized by alternating stacking of adenine and benzoyl residues of the neighboring molecules in the crystal lattice. The conformation of the 5'-hydroxymethylene in 3'-O- anthraniloyladenosine is gauche-gauche. There are two intramolecular and two intermolecular hydrogen bonds and several H-bridges with surrounding water molecules. The predominant structure of 3'-O-anthraniloyladenosine in solution, as determined by NMR spectroscopy, is 2'-endo,gauche-gauche and anti for the sugar ring pucker, the torsion angle around the C4'-C5'bond and the torsion angle around the C1'-N9 bond, respectively. The 2'-endo conformation of the ribose in 2'(3')-O-aminoacyladenosine, which places the adenine and aminoacyl residues in equatorial and axial positions, respectively, could serve as a structural element that is recognized by enzymes that interact with aminoacyl-tRNA or by ribosomes to differentiate between aminoacylated and non-aminoacylated tRNA. 相似文献
20.
Honglue Shi Mary C. Clay Atul Rangadurai Bharathwaj Sathyamoorthy David A. Case Hashim M. Al-Hashimi 《Journal of biomolecular NMR》2018,70(4):229-244
NMR relaxation dispersion studies indicate that in canonical duplex DNA, Watson–Crick base pairs (bps) exist in dynamic equilibrium with short-lived low abundance excited state Hoogsteen bps. N1-methylated adenine (m1A) and guanine (m1G) are naturally occurring forms of damage that stabilize Hoogsteen bps in duplex DNA. NMR dynamic ensembles of DNA duplexes with m1A–T Hoogsteen bps reveal significant changes in sugar pucker and backbone angles in and around the Hoogsteen bp, as well as kinking of the duplex towards the major groove. Whether these structural changes also occur upon forming excited state Hoogsteen bps in unmodified duplexes remains to be established because prior relaxation dispersion probes provided limited information regarding the sugar-backbone conformation. Here, we demonstrate measurements of C3′ and C4′ spin relaxation in the rotating frame (R1ρ) in uniformly 13C/15N labeled DNA as sensitive probes of the sugar-backbone conformation in DNA excited states. The chemical shifts, combined with structure-based predictions using an automated fragmentation quantum mechanics/molecular mechanics method, show that the dynamic ensemble of DNA duplexes containing m1A–T Hoogsteen bps accurately model the excited state Hoogsteen conformation in two different sequence contexts. Formation of excited state A–T Hoogsteen bps is accompanied by changes in sugar-backbone conformation that allow the flipped syn adenine to form hydrogen-bonds with its partner thymine and this in turn results in overall kinking of the DNA toward the major groove. Results support the assignment of Hoogsteen bps as the excited state observed in canonical duplex DNA, provide an atomic view of DNA dynamics linked to formation of Hoogsteen bps, and lay the groundwork for a potentially general strategy for solving structures of nucleic acid excited states. 相似文献