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1.
Drug binding by branched DNA molecules: analysis by chemical footprinting of intercalation into an immobile junction 总被引:3,自引:0,他引:3
Branched DNA structures interact with drugs differently from unbranched control duplexes of similar sequence. A specific interaction between the reagent (methidiumpropyl-EDTA).Fe(II) [MPE.Fe(II)] and a branched DNA molecule formed from 16-mer oligonucleotide strands has been reported [Guo, Q., Seeman, N. C., & Kallenbach, N. R. (1989) Biochemistry 28, 2355-2359]. The structure of the branched molecule is thought to be made up of two double-helical stacking domains with an overall twofold symmetry across the branch site. The MPE-Fe(II) interaction occurs predominantly at or adjacent to the branch site and is eliminated by a second intercalator, propidium iodide. Further studies on the nature and properties of this site are presented here. Comparison of the patterns of scission of linear duplex and branched tetramer by EDTA.Fe(II), MPE.Fe(II), and Cu(I)-(o-phenanthroline)2 [(OP)2Cu(I)] provides a higher resolution picture of the site of enhanced binding. In particular, the sensitive footprinting afforded by (OP)2Cu(I) allows us to localize the major site of preferential interaction with propidium precisely to the branch point itself, with a roughly twofold symmetric pattern of cuts resulting. In detail, the differential pattern with respect to each duplex control is distinct for each arm of the junction. Excess propidium results in apparent reversal of the crossover isomer of the junction, indicating a possible additional avenue for the action of drugs in biological systems--effects on the products of recombination. 相似文献
2.
Characterization of a bimobile DNA junction 总被引:1,自引:0,他引:1
M Lu Q Guo J E Mueller B Kemper F W Studier N C Seeman N R Kallenbach 《The Journal of biological chemistry》1990,265(28):16778-16785
We present here a chemical and enzymatic footprinting analysis of a branched DNA molecule formed from four complementary 50-mer strands. These strands are designed to form a stable junction, in which two steps of branch point migration freedom are possible. Exposure of the junction to Fe(II).EDTA shows protection of 3 or 4 residues in each strand at the branch, while two resolvase enzymes (endonuclease VII from phage T4 and endonuclease I from phage T7), cleave all four strand near the branch. Chemical footprinting of this junction using the reagents MPE.Fe(II) and (OP)2Cu(I) shows that the branch site is hyper-reactive to cutting induced by these probes as it is in an immobile four-arm junction. The effects involve more residues than in the immobile case. In the absence of divalent cations, the structure of the junction alters, sites of enhanced cleavage by MPE.Fe(II) and (OP)2Cu(I) disappear, and purines at the branch become reactive to diethyl pyrocarbonate. Our interpretation of these results is based on the properties of immobile junction analogs and their response to these probes. In the presence of Mg2+, the three migrational isomers coexist, each probably in the form of a 2-fold symmetric structure with two helical arms stacked. 相似文献
3.
Asymmetric structure of a three-arm DNA junction 总被引:6,自引:0,他引:6
We present here experimental evidence that three-arm branched DNA molecules form an asymmetric structure in the presence of Mg2+. Electrophoretic mobility and chemical and enzymatic footprinting experiments on a three-arm branched DNA molecule formed from three 16-mer strands are described. The electrophoretic mobilities of three species of a three-arm junction in which pairs of arms are extended are found to differ in the presence of Mg2+: one combination of elongated arms migrates significantly faster than the other two. This effect is eliminated in the absence of Mg2+, leading us to suggest that the three-arm DNA junction forms an asymmetric structure due to preferential stacking of two of the arms at the junction in the presence of Mg2+. The pattern of self-protection of each 16-mer strand of the core complex exposed to Fe(II).EDTA and DNase I scission is unique, consistent with formation of an asymmetric structure in the presence of Mg2+. We conclude that three-arm junctions resemble four-arm junctions in showing preferential stacking effects at the branch site. Comparison of the scission patterns of linear duplexes and the branched trimer by the reactive probes methidiumpropyl-EDTA.Fe(II) [MPE.Fe(II)] and Cu(I)-[o-phenanthroline]2 [(OP)2CuI] further indicates that the branch point represents a site of enhanced binding for drugs, as it does in the four-arm case. Reaction with diethyl pyrocarbonate (DEPC), a purine-specific probe sensitive to conformation, is enhanced at the branch site, consistent with loosening of base pairing or unpairing at this point.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
4.
Kaluarachchi H Siebel JF Kaluarachchi-Duffy S Krecisz S Sutherland DE Stillman MJ Zamble DB 《Biochemistry》2011,50(49):10666-10677
SlyD is a Ni(II)-binding protein that contributes to nickel homeostasis in Escherichia coli. The C-terminal domain of SlyD contains a rich variety of metal-binding amino acids, suggesting broader metal binding capabilities, and previous work demonstrated that the protein can coordinate several types of first-row transition metals. However, the binding of SlyD to metals other than Ni(II) has not been previously characterized. To improve our understanding of the in vitro metal-binding activity of SlyD and how it correlates with the in vivo function of this protein, the interactions between SlyD and the series of biologically relevant transition metals [Mn(II), Fe(II), Co(II), Cu(I), and Zn(II)] were examined by using a combination of optical spectroscopy and mass spectrometry. Binding of SlyD to Mn(II) or Fe(II) ions was not detected, but the protein coordinates multiple ions of Co(II), Zn(II), and Cu(I) with appreciable affinity (K(D) values in or below the nanomolar range), highlighting the promiscuous nature of this protein. The order of affinities of SlyD for the metals examined is as follows: Mn(II) and Fe(II) < Co(II) < Ni(II) ~ Zn(II) ? Cu(I). Although the purified protein is unable to overcome the large thermodynamic preference for Cu(I) and exclude Zn(II) chelation in the presence of Ni(II), in vivo studies reveal a Ni(II)-specific function for the protein. Furthermore, these latter experiments support a specific role for SlyD as a [NiFe]-hydrogenase enzyme maturation factor. The implications of the divergence between the metal selectivity of SlyD in vitro and the specific activity in vivo are discussed. 相似文献
5.
D H Tjahjono S Mima T Akutsu N Yoshioka H Inoue 《Journal of inorganic biochemistry》2001,85(2-3):219-228
The interaction of transition metal complexes of cationic porphyrins bearing five membered rings, meso-tetrakis(1,2-dimethylpyrazolium-4-yl)porphyrin (MPzP, M=Mn(III), Ni(II), Cu(II) or Zn(II)), with calf thymus DNA (ctDNA) has been studied. Metalloporphyrins NiPzP and CuPzP are intercalated into the 5'GC3' step of ctDNA. MnPzP is bound edge-on at the 5'TA3' step of the minor groove of ctDNA, while ZnPzP is bound face-on at the 5'TA3' step of the major groove of ctDNA. The binding constants of the metalloporphyrins to ctDNA range from 1.05x10(5) to 2.66x10(6) M(-1) and are comparable to those of other reported cationic porphyrins. The binding process of the metallopyrazoliumylporphyrins to ctDNA is endothermic and entropically driven. These results have revealed that the kind of central metal ions of metalloporphyrins influences the binding characteristics of the porphyrin to DNA. 相似文献
6.
Drug binding by branched DNA: selective interaction of the dye stains-all with an immobile junction 总被引:1,自引:0,他引:1
The thiacarbocyanine dye Stains-All (4,5:4',5'-dibenzo-3,3'-diethyl-9-methylthiacarbocyanine bromide) is one of a large number of cyanine dyes introduced as photosensitizers in the photographic industry. Stains-All is used in histology as a stain for nucleic acids, proteins, polysaccharides, and lipids. We report here that the dye colors branched DNA molecules differently from linear duplexes and use footprinting experiments with methidiumpropyl-EDTA-Fe(II) [MPE.Fe(II)] and bis(o-phenanthroline)copper(I) [(O-P)2Cu(I)] to show that Stains-All interacts preferentially at the branch point of a four-arm DNA structure. A titration experiment allows us to estimate that the interaction of the dye with the branch has a dissociation constant below 45 nM, tighter than that of ethidium or methidium by over 2 orders of magnitude. Probing the interaction with the purine-specific reagent diethyl pyrocarbonate (DEPC) implies that the dye induces an asymmetric distortion near the branch in the major grooves of double helix in the junction. 相似文献
7.
Dalyan YB Haroutiunian SG Ananyan GV Vardanyan VI Lando DY Madakyan VN Kazaryan RK Messory L Orioli P Benight AS 《Journal of biomolecular structure & dynamics》2001,18(5):677-687
Interactions of meso-tetra-(4-N-oxyethylpyridyl) porphyrin (TOEPyP(4)), its 3-N analog (TOEPyP(3)) and their Co, Cu, Ni, Zn metallocomplexes with duplex DNA have been investigated by uv/visible absorbance and circular dichrosim spectroscopies. Results reveal the interactions of these complexes with duplex DNA are of two types. (1) External binding of duplex DNA by metalloporphyrins containing Zn and Co, and (2) Binding of duplex DNA both externally and internally (by intercalation) by porphyrins not containing metals, and metalloporphyrins containing Cu and Ni. Results indicate that (4N-oxyethylpyridyl) porphyrins intercalate more preferably in the structure of duplex DNA and have weaker external binding than 3N-porphyrins. 相似文献
8.
Yeva B. Dalyan Samvel G. Haroutiunian Gayane V. Ananyan Volodya I. Vardanyan Dmitri Y. Lando Valeri N. Madakyan 《Journal of biomolecular structure & dynamics》2013,31(5):677-687
Abstract Interactions of meso-tetra-(4-N-oxyethylpyridyl) porphyrin (TOEPyP(4)), its 3-N analog (TOEPyP(3)) and their Co, Cu, Ni, Zn metallocomplexes with duplex DNA have been investigated by uv/visible absorbance and circular dichrosim spectroscopies. Results reveal the interactions of these complexes with duplex DNA are of two types. (1) External binding of duplex DNA by metalloporphyrins containing Zn and Co, and (2) Binding of duplex DNA both externally and internally (by intercalation) by porphyrins not containing metals, and metalloporphyrins containing Cu and Ni. Results indicate that (4N-oxyethylpyridyl) porphyrins intercalate more preferably in the structure of duplex DNA and have weaker external binding than 3N-porphyrins. 相似文献
9.
Two Holliday junction analogs, JA and JP, containing identical base-paired arms have been constructed from oligonucleotides. The former is constrained to adopt an antiparallel Sigal-Alberts structure, and the latter a parallel structure, by means of single strand d(T)9 tethers. We evaluate here the free energy difference between JA and JP using two different methods. One is a direct measurement of the ratio of the equilibrium constants for formation of branched structures from intact duplexes using one labeled strand and a competition assay. The second method estimates the difference in stability from the difference in thermal denaturation temperatures of JA and JP, using urea to shift the tm of the complexes. Both methods reveal a small free energy difference between the two complexes: JA is more stable than JP by -1.1(+/- 0.4) kcal (mol junction)-1, at 25 degrees C, 5 mM-Mg2+, from the first method, and by -1.6(+/- 0.3) kcal (mol junction)-1, according to the second. DNase I and the resolvase, endonuclease I from phage T7, cleave JA differently from JP in the vicinity of the branch, indicating that the structures of these two models differ at this site. Diethyl pyrocarbonate also reveals a difference in the major grooves. Comparison of the scission patterns of JA and JP by the reactive chemical probes methidium-propyl-EDTA..Fe(II), [MPE.Fe(II)] and Cu(I)-[o-phenanthroline]2,[(OP)2Cu(I)], indicates that in both cases the branch point is a site of enhanced binding for drugs, as it is in the untethered four-arm junction containing the same core sequence at the branch. 相似文献
10.
A new ligand N-salicyloyl-N'-o-hydroxythiobenzhydrazide (H2Sotbh) forms complexes [Mn(HSotbh)2], [Fe(Sotbh-H)(H2O)2], [M(Sotbh)] [M=Co(II), Cu(II) and Zn(II)] and [Ni(Sotbh)(H(2)O)2], which were characterized by various physico-chemical techniques. M?ssbauer spectrum of [Fe(Sotbh-H)(H2O)2] reveals the quantum admixture of 5/2 and 3/2 spin-states. Mn(II), Cu(II) and Ni(II) complexes were observed to inhibit the growth of tumor in vitro, whereas, Fe(III), Co(II), Zn(II) complexes did not. In vivo administration of Mn(II), Cu(II) and Ni(II) resulted in prolongation of survival of tumor bearing mice. Tumor bearing mice administered with Mn(II), Cu(II) and Ni(II) complexes showed reversal of tumor growth associated induction of apoptosis in lymphocytes. The paper discusses the possible mechanisms and therapeutic implication of the H2Sotbh and its metal complexes in tumor regression and tumor growth associated immunosuppression. 相似文献
11.
The ability of several metals to inhibit dopamine beta-monooxygenase was measured and compared with their ability to compete with the binding of 64Cu to the water-soluble form of the bovine adrenal enzyme at pH 6.0. In the presence of an optimal concentration of copper (0.5 microM in the present assay system), an inhibition was observed upon addition of Hg(II), Zn(II), or Ni(II). Only a small fraction of the inhibition with these metals may be due to uncoupling of electron transport from hydroxylation. Preincubation of these metals with the Cu-depleted apoenzyme before addition of copper, revealed a stronger inhibition than if copper was added before the other metals. Hg(II), Zn(II), and Ni(II) also compete with the binding of 64Cu(II) to the protein. Hg(II) was the most effective and Ni(II) the least effective of these metals, both with respect to inhibition of the enzyme activity and to prevent the binding of 64Cu(II). Competition experiments on the binding of Zn(II) and 64Cu in the presence and absence of ascorbate, indicated i) a similar affinity of Cu(I) and Cu(II) to the native enzyme, and ii) a more rapid binding of Cu(I) than Cu(II) to the Cu-depleted and Zn-containing enzyme. Al(III), Fe(II), Mg(II), Mn(II), Co(II), Cd(II), and Pb(II) neither inhibited the enzyme activity nor competed with the binding of 64Cu(II) to the protein (Fe(II) was not tested for binding). Of those metals cited above only Cu(II)/Cu(I) was able to reactivate the apoenzyme. 相似文献
12.
Choi DW Do YS Zea CJ McEllistrem MT Lee SW Semrau JD Pohl NL Kisting CJ Scardino LL Hartsel SC Boyd ES Geesey GG Riedel TP Shafe PH Kranski KA Tritsch JR Antholine WE DiSpirito AA 《Journal of inorganic biochemistry》2006,100(12):2150-2161
Methanobactin (mb) is a novel chromopeptide that appears to function as the extracellular component of a copper acquisition system in methanotrophic bacteria. To examine this potential physiological role, and to distinguish it from iron binding siderophores, the spectral (UV–visible absorption, circular dichroism, fluorescence, and X-ray photoelectron) and thermodynamic properties of metal binding by mb were examined. In the absence of Cu(II) or Cu(I), mb will bind Ag(I), Au(III), Co(II), Cd(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(VI), or Zn(II), but not Ba(II), Ca(II), La(II), Mg(II), and Sr(II). The results suggest metals such as Ag(I), Au(III), Hg(II), Pb(II) and possibly U(VI) are bound by a mechanism similar to Cu, whereas the coordination of Co(II), Cd(II), Fe(III), Mn(II), Ni(II) and Zn(II) by mb differs from Cu(II). Consistent with its role as a copper-binding compound or chalkophore, the binding constants of all the metals examined were less than those observed with Cu(II) and copper displaced other metals except Ag(I) and Au(III) bound to mb. However, the binding of different metals by mb suggests that methanotrophic activity also may play a role in either the solubilization or immobilization of many metals in situ. 相似文献
13.
Interactions of porphyrins with nucleic acids 总被引:24,自引:0,他引:24
The interactions of nucleic acids with water-soluble porphyrins and metalloporphyrins have been investigated by stopped-flow and temperature-jump techniques. Both natural DNA (calf thymus) and synthetic homopolymers [poly(dG-dC) and poly(dA-dT)] have been employed. The porphyrins studied belong to the tetrakis(4-N-methylpyridyl)porphine (H2TMpyP-4) series and can be divided into two groups: (i) those which have no axial ligands when bound to nucleic acids [e.g., Ni(II), Cu(II), and the nonmetallic derivatives] and (ii) those which maintain axial ligands upon binding [e.g., Mn(III), Fe(III), Co(III), and Zn(II) derivatives]. The reaction of both axially and nonaxially liganded porphyrins at AT sites is too rapid to be measured by the kinetic methods utilized, whereas at GC sites the interaction of the nonaxially liganded porphyrins is in the millisecond time range and can be monitored by both stopped-flow and temperature-jump techniques. These results corroborate previous static studies, utilizing visible spectroscopy and circular dichroism, which indicate that the formation of an intercalated complex occurs only at GC base pair sites with porphyrins which do not possess axial ligands. With all the porphyrins investigated, the complexes formed at AT sites are envisioned as being of an "external" type involving some degree of overlap between the porphyrin and the bases of the duplex. In relaxation experiments of poly-(dG-dC) with H2TMpyP-4, a large, reproducible effect is observed which can be analyzed as a single exponential. Rate constants for association and dissociation of the H2TMpyP-4/poly(dG-dC) complex are 3.7 X 10(5) M-1 s-1 and 1.8 s-1, respectively. Relaxation studies of mixtures of poly(dA-dT) and poly(dG-dC) with H2TMpyP-4 indicate that the transfer of the porphyrin from one homopolymer to another occurs via a mechanism involving dissociation rather than direct transfer.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
14.
Dong W. Choi Young S. Do Corbin J. Zea Marcus T. McEllistrem Sung-W. Lee Jeremy D. Semrau Nicola L. Pohl Clint J. Kisting Lori L. Scardino Scott C. Hartsel Eric S. Boyd Gill G. Geesey Theran P. Riedel Peter H. Shafe Kim A. Kranski John R. Tritsch William E. Antholine Alan A. DiSpirito 《Journal of inorganic biochemistry》2006,100(12):2150
Methanobactin (mb) is a novel chromopeptide that appears to function as the extracellular component of a copper acquisition system in methanotrophic bacteria. To examine this potential physiological role, and to distinguish it from iron binding siderophores, the spectral (UV–visible absorption, circular dichroism, fluorescence, and X-ray photoelectron) and thermodynamic properties of metal binding by mb were examined. In the absence of Cu(II) or Cu(I), mb will bind Ag(I), Au(III), Co(II), Cd(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(VI), or Zn(II), but not Ba(II), Ca(II), La(II), Mg(II), and Sr(II). The results suggest metals such as Ag(I), Au(III), Hg(II), Pb(II) and possibly U(VI) are bound by a mechanism similar to Cu, whereas the coordination of Co(II), Cd(II), Fe(III), Mn(II), Ni(II) and Zn(II) by mb differs from Cu(II). Consistent with its role as a copper-binding compound or chalkophore, the binding constants of all the metals examined were less than those observed with Cu(II) and copper displaced other metals except Ag(I) and Au(III) bound to mb. However, the binding of different metals by mb suggests that methanotrophic activity also may play a role in either the solubilization or immobilization of many metals in situ. 相似文献
15.
Anitha M. Thomas 《Inorganica chimica acta》2004,357(8):2315-2323
New ternary copper(II) complexes of formulations [Cu(Ph-tsc)B] (B=1,10-phenanthroline, phen (1); dipyridoquinoxaline, dpq (2); dipyridophenazine, dppz (3); Ph-H2tsc, salicylaldehyde-N(4)-phenylthiosemicarbazone) and [Cu(Me-tsc)(phen)] (4, Me-H2tsc, salicylaldehyde-N(4)-methylthiosemicarbazone) are prepared, and their DNA binding and cleavage properties studied. Complex 1 has been characterized by single crystal X-ray crystallography. The molecular structure shows a distorted square pyramidal (4 + 1) geometry of the complex with the dianionic NSO-donor N(4)-phenyl-substituted thiosemicarbazone binding at the basal plane and the NN-donor planar heterocyclic base (phen) displaying axial-equatorial coordination. The one-electron paramagnetic complexes exhibit axial EPR spectra and show a d-d band near 580 nm for the phen and near 720 nm for the dpq, dppz complexes in their electronic spectra in DMF. The complexes show quasireversible cyclic voltammetric response near 0.08 V vs. SCE in DMF-0.1 M TBAP assignable to the Cu(II)/Cu(I) couple. The Ph-tsc complexes display good binding propensity to calf thymus (CT) DNA. They also show oxidative cleavage of supercoiled (SC) pUC19 DNA in dark under aerobic condition in the presence of mercaptopropionic acid. The complexes exhibit light-induced DNA cleavage activity at 312 and 532 nm. Mechanistic investigations reveal DNA minor groove binding for the phen and dpq complexes, and major groove binding for the dppz species. The complexes are cleavage inactive under argon atmosphere. In the ternary structure, the thiosemicarbazones, dpq and dppz act as photosensitizers, while the planar heterocyclic bases are binder to DNA. The mechanistic pathways involved and the role of metal in the DNA cleavage reactions are discussed. 相似文献
16.
Summary Cabbage plants were grown for 55 days with a nutrient solution containing 1 and 10 ppm of V, Cr(III), Cr(VI), Mn, Fe, Co, Ni, Cu, Zn, Cd, Hg(I), orHg(II). A comparison of the plant growth and chemical analysis revealed that Cr(VI), Cu, Cd, and Hg(II) in the solution are most toxic to the plant growth (hence detrimental to the cabbage-head formation) and Mn, Fe, and Zn are less toxic than other heavy metals, and that Mn, Zn, Co, Ni, and Cd and translocated into all the plant organs while V, Cr(III), Cr(VI), Fe, Cu, Hg(I), and Hg(II) are accumulated in the roots. 相似文献
17.
A. Kosiha C. Parthiban Samuele Ciattini Laura Chelazzi 《Journal of biomolecular structure & dynamics》2013,31(16):4170-4181
Protein binding, DNA binding/cleavage and in vitro cytotoxicity studies of 2-((3-(dimethylamino)propyl)amino)naphthalene-1,4-dione (L) and its four coordinated M(II) complexes [M(II) = Co(II), Cu(II), Ni(II) and Zn(II)] have been investigated using various spectral techniques. The structure of the ligand was confirmed by spectral and single crystal XRD studies. The geometry of the complexes has been established using analytical and spectral investigations. These complexes show good binding tendency to bovine serum albumin (BSA) exhibiting high binding constant values (105 M?1) when compared to free ligand. Fluorescence titration studies reveal that these compounds bind strongly with CT-DNA through intercalative mode (Kapp 105 M?1) and follow the order: Cu(II) > Zn(II) > Ni(II) > Co(II) > L. Molecular docking study substantiate the strength and mode of binding of these compounds with DNA. All the complexes efficiently cleaved pUC18-DNA via hydroxyl radical mechanism and the Cu(II) complex degraded the DNA completely by converting supercoiled form to linear form. The complexes demonstrate a comparable in vitro cytotoxic activity against two human cancer cell lines (MCF-7 and A-549), which is comparable with that of cisplatin. AO/EB and DAPI staining studies suggest apoptotic mode of cell death, in these cancer cells, with the compounds under investigation. 相似文献
18.
Gossypol, a polyphenolic binaphthyl dialdehyde found in cotton seeds, is a dietary mutagen and a potential male contraceptive. In the presence of Cu(II), gossypol caused breakage of supercoiled plasmid pBR322 DNA. The products were relaxed circles or a mixture of these and linear molecules. Other metal ions tested [Ni(II), Co(II), Mn(II), and Fe(II)] were ineffective or less effective in the DNA breakage reaction. In the case of gossypol-Cu(II) mediated cleavage, (Cu(I) was shown to be an essential intermediate by using the Cud) sequestering reagent bathocuproine. By using job plots, it was established that in the absence of DNA, eight Cu(II) ions can be reduced by one gossypol molecule. The involvement of active oxygen species, such as singlet oxygen and H2O2, was established by the inhibition of DNA breakage by catalase and by sodium azide. It was further shown that gossypol is capable of directly producing H2O2. 相似文献
19.
The Rap protein of phage lambda is an endonuclease that nicks branched DNA structures. It has been proposed that Rap can nick D-loops formed during phage recombination to generate splice products without the need for the formation of a 4-strand (Holliday) junction. The structure specificity of Rap was investigated using a variety of branched DNA molecules made by annealing partially complementary oligo-nucleotides. On Holliday junctions, Rap endonuclease shows a requirement for magnesium or manganese ions, with Mn(2+)supporting 5-fold more cleavage than Mg(2+). The location of endonuclease incisions was determined on 3'-tailed D-loop, bubble, flayed duplex, 5'-flap and Y junction DNA substrates. In all cases, Rap preferentially cleaves at the branch point of these molecules. With a flayed duplex, incisions are made in the duplex adjacent to the single-strand arms. Comparison of binding and cleavage specificities revealed that Rap is highly structure-specific and exhibits a clear preference for 4- and 3-stranded DNA over Y and flayed duplex DNA. Almost no binding or cleavage was detected with duplex, partial duplex and single-stranded DNA. Thus Rap endonuclease shows a bias for structures that resemble D-loop and Holliday junction recombination intermediates. 相似文献
20.
Resonance Raman, NMR, and visible spectroscopies, as well as viscosity and equilibrium dialysis studies were used to assess the effect of the N-alkyl substituent of meso-tetrakis(4-N-alkylpyridinium-4-yl)porphyrin cations on DNA binding. The DNAs studied include the native DNA, calf thymus DNA (CT DNA), the synthetic polynucleotides [poly(dGdC)]2 and [poly(dAdT)]2, and the oligonucleotide d(TATACGTATA)2. Both the porphyrins and the metalloporphyrins containing Ni(II) were examined with the N-alkyl = propyl (TPrpyP(4) and NiTPrpyP(4)) and 2-hydroxyethyl (TEtOHpyP(4) and NiTEtOHpyP(4)). The results were compared to those from the parent porphyrins with the N-methyl substituent (TMpyP(4) and NiTMpyP(4)). For almost all the comparisons made, the new porphyrin cations gave results very similar to those for the TMpyP(4) species. The resonance Raman study indicated that for the three DNA polymers all the Ni species were in the four-coordinate form when bound to all three polymers. It is suggested that both TPrpyP(4) and TEtOHpyP(4) bind to GC regions of DNA in the same intercalative manner as TMpyP(4) with the N-alkyl substituent extended into the solvent. For AT regions of DNA, the binding of TPrpyP(4) and TEtOHpyP(4) is nonintercalative, as found previously for TMpyP(4). The NiPrpy(4) and NiTEtOHpyP(4) cations bind to these polymers in a similar manner to the apo-porphyrins. The similar Raman spectral changes for the three Ni porphyrins upon addition of [poly(dAdT)]2 suggest that partial intercalation is not occurring because models indicate that it would be difficult to accommodate the bulkier N-alkyl substituents. 相似文献