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1.
Formation and stability of repairable pyrimidine photohydrates in DNA   总被引:4,自引:0,他引:4  
Ultraviolet irradiation of poly(dG-dC) and poly(dA-dU) in solution produces pyrimidine hydrates that are repaired by bacterial and mammalian DNA glycosylases [Boorstein et al. (1989) Biochemistry 28, 6164-6170]. Escherichia coli endonuclease III was used to quantitate the formation and stability of these hydrates in the double-stranded alternating copolymers poly(dG-dC) and poly(dA-dU). When poly(dG-dC) was irradiated with 100 kJ/m2 of 254-nm light at pH 8.0, 2.2% of the cytosine residues were converted to cytosine hydrate (6-hydroxy-5,6-dihydrocytosine) while 0.09% were converted to uracil hydrate (6-hydroxy-5,6-dihydrouracil). To measure the stability of these products, poly(dG-dC) was incubated in solution for up to 24 h after UV irradiation. Cytosine hydrate was stable at 4 degrees C and decayed at 25, 37, and 55 degrees C with half-lives of 75, 25, and 6 h. Uracil hydrate produced in irradiated poly(dA-dU) was stable at 4 degrees C and at 25 degrees C and decayed with a half-life of 6 h at 37 degrees C and less than 0.5 h at 55 degrees C. Uracil hydrate and uracil were also formed in irradiated poly(dG-dC). These experiments demonstrate that UV-induced cytosine hydrate may persist in DNA for prolonged time periods and also undergo deamination to uracil hydrate, which in turn undergoes dehydration to yield uracil. The formation and stability of these photoproducts in DNA may have promoted the evolutionary development of the repair enzyme endonuclease III and analogous DNA glycosylase/endonuclease activities of higher organisms, as well as the development of uracil-DNA glycosylase.  相似文献   

2.
Cytosine glycols (5,6-dihydroxy-5,6-dihydrocytosine) are initial products of cytosine oxidation. Because these products are not stable, virtually all biological studies have focused on the stable oxidation products of cytosine, including 5-hydroxycytosine, uracil glycols and 5-hydroxyuracil. Previously, we reported that the lifetime of cytosine glycols was greatly enhanced in double-stranded DNA, thus implicating these products in DNA repair and mutagenesis. In the present work, cytosine and uracil glycols were generated in double-stranded alternating co-polymers by oxidation with KMnO4. The half-life of cytosine glycols in poly(dG-dC) was 6.5 h giving a ratio of dehydration to deamination of 5:1. At high substrate concentrations, the excision of cytosine glycols from poly(dG-dC) by purified endonuclease III was comparable to that of uracil glycols, whereas the excision of these substrates was 5-fold greater than that of 5-hydroxycytosine. Kinetic studies revealed that the Vmax was several fold higher for the excision of cytosine glycols compared to 5-hydroxycytosine. In contrast to cytosine glycols, uracil glycols did not undergo detectable dehydration to 5-hydroxyuracil. Replacing poly(dG-dC) for poly(dI-dC) gave similar results with respect to the lifetime and excision of cytosine glycols. This work demonstrates the formation of cytosine glycols in DNA and their removal by base excision repair.  相似文献   

3.
Hydrolytic damages to DNA can occur at physiological conditions. The possible role of DNA conformation on the distribution of such alterations of pyrimidines was investigated. Model compounds used were the synthetic alternating copolymer poly(dG-dC):poly(dG-dC) and the homopolymer poly(dG):poly(dC). Base damages were assayed by paper chromatography using polymers radioactively labeled in cytosine. Conformational changes were assayed by circular dichroic spectral changes. Incubation and heating of the polymers in 1 mM MnCl2 caused the spectral shift reported for the left-handed Z-DNA conformation in the alternating copolymer and the change reported for the triple helix in the homopolymer. After incubation in 85°C, incidences of base damages were compared between the polymers. The presence of manganese reduced depyrimidination in both polymers. Rates of cytosine deamination to uracil were substantial and did not vary among the various conformational states.  相似文献   

4.
Pyrimidine hydrates are products of ultraviolet irradiation of DNA. We have already demonstrated the formation of both cis-thymine hydrate and trans-thymine hydrate (6-hydroxy-5,6-dihydrothymine) in irradiated poly(dA-dT):poly(dA-dT). These are released from DNA as free bases by bacterial or human glycosylases. Thymine hydrate stabilities were studied in irradiated DNA substrates using purified E. coli endonuclease III as a reagent for their removal. After irradiation, substrate poly(dA-dT):poly(dA-dT), radiolabeled in thymine, was incubated at 50, 60, 70 or 80 degrees C, cooled, and then reacted with the enzyme under standard conditions. Thymine hydrates were assayed by enzymic release of labeled material into the ethanol-soluble fraction. Their identities were confirmed by high performance liquid chromatography. The decay of thymine hydrates in heated DNA followed first-order kinetics with a k = 2.8 x 10(-5)/sec at 80 degrees C. These hydrates were also detected in lesser quantities in the unirradiated, control substrate. Extrapolation from an Arrhenius plot yields an estimated half-life of 33.3 hours at 37 degrees C for DNA thymine hydrates. Such stability, together with their formation in unirradiated DNA, suggest thymine hydrates to be formed under physiological conditions and to be sufficiently stable in DNA to be potentially genotoxic. This necessitates their constant removal from DNA by the excision-repair system.  相似文献   

5.
Ultraviolet irradiation of DNA results in various pyrimidine modifications. We have demonstrated formation of both cis-thymine hydrate and trans-thymine hydrate (6-hydroxy-5,6-dihydrothymine) in UV-irradiated poly(dA-dT):poly(dA-dT). Both are released from DNA as free bases by bacterial and human glycosylases. Thymine hydrates are stable in DNA and can be detected in control, unirradiated substrates. We examined the effects of thymine hydrates in UV-irradiated substrate poly(dA-dT):poly(dA-dT) on E. coli DNA polymerase I activity. Enzymic incorporation of labeled thymidine-5'-monophosphate significantly decreased with increasing UV dose. Reversal of DNA thymine hydrates to thymines by mild heating of the substrate prior to enzymic reaction resulted in partial recovery of nucleotide incorporation. Cyclobutane thymine dimers are formed between non-adjacent thymines in UV-irradiated poly(dA-dT):poly(dA-dT). These are responsible for the incomplete recovery of DNA polymerase activity following heating due to their heat stability. Analyses of the irradiated and hydrolyzed substrate also demonstrated formation of minor yields of photoproducts formed by covalent linkage of adjacent thymines and adenines by UV-irradiation. Therefore, the thymine hydrates formed in UV-irradiated DNA partially inhibit polymerase activity during DNA synthesis and thus could be potentially lethal if unrepaired.  相似文献   

6.
The acridine dye quinacrine and its interactions with calf thymus DNA, poly(dA-dT) · poly (dA-dT), and poly (dG-dC) · poly(dG-dC) were studied by light absorption, linear dichroism, and fluorescence spectroscopy. The transition moments of quinacrine give rise to absorption bands polarized along the short axis (400–480-nm band), and the long axis (345-nm and 290-nm bands) of the molecule, respectively. Linear dichroism studies show that quinacrine intercalates into calf thymus DNA as well as into the polynucleotides, displaying fairly homogeneous binding to poly (dA-dT) · poly (dA-dT), but more than one type of intercalation site for calf thymus DNA and poly (dG-dC) · poly(dG-dC). Fluorescence spectroscopy shows that for free quinacrine the pK = 8.1 between the mono- and diprotonated states also remains unchanged in the excited state. Quinacrine bound to calf thymus DNA and polynucleotides exhibits light absorption typical for the intercalated diprotonated form. The fluorescence enhancement of quinacrine bound to poly (dA-dT) · poly(dA-dT) may be due to shielding from water interactions involving transient H-bond formation. The fluorescence quenching in poly(dG-dC) · poly(dG-dC) may be due to excited state electron transfer from guanine to quinacrine. © 1993 John Wiley & Sons, Inc.  相似文献   

7.
Interaction of topotecan (TPT) with synthetic double-stranded polydeoxyribonucleotides has been studied in solutions of low ionic strength at pH = 6.8 by linear flow dichroism (LD), circular dichroism (CD), UV-Vis absorption and Raman spectroscopy. The complexes of TPT with poly(dG-dC).poly(dG-dC), poly(dG).poly(dC), poly(dA-dC).poly(dG-dT), poly(dA).poly(dT) and previously studied by us complexes of TPT with calf thymus DNA and coliphage T4 DNA have been shown to have negative LD in the long-wavelength absorption band of TPT, whereas the complex of TPT with poly(dA-dT).poly(dA-dT) has positive LD in this absorption band of TPT. Thus, there are two different types of TPT complexes with the polymers. TPT has been established to bind preferably to GC base pairs because its affinity to the polymers of different GC composition decreases in the following order: poly(dG-dC).poly(dG-dC) > poly(dG).poly(dC) > poly(dA-dC).poly(dG-dT) > poly(dA).poly(dT). The presence of DNA has been shown to shift monomer-dimer equilibrium in TPT solutions toward dimer formation. Several duplexes of the synthetic polynucleotides bound together by the bridges of TPT dimers may participate in the formation of the studied type of TPT-polynucleotide complexes. Molecular models of TPT complex with linear and ring supercoiled DNAs and with deoxyguanosine have been considered. TPT (and presumably all camptothecin family) proved to be a representative of a new class of DNA-specific ligands whose biological action is associated with formation of dimeric bridges between two DNA duplexes.  相似文献   

8.
B Malfoy  B Hartmann    M Leng 《Nucleic acids research》1981,9(21):5659-5669
Poly(dG-dC) . poly(dG-dC) was modified by chlorodiethylenetriamino platinum (II) chloride, cis-dichlorodiammine platinum (II) and trans-dichlorodiammine platinum (II), respectively. The conformation of these modified poly(dG-dC) . poly(dG-dC) was studied by circular dichroism. In 4 M Na+, the circular dichroism spectra of poly(dG-dC)dien-Pt (0 less than or equal to rb less than or equal to 0.2) are similar (rb is the amount of bound platinum per base). It is concluded that the conformation of these polymers belongs to the Z-family. Dien-Pt complexes stabilize the Z-form. The midpoint of the Z goes to B transition of poly(dG-dC)dien-Pt(0.12) is at 0.2 M NaCl. Moreover another B goes to Z transition is observed at lower salt concentration (midpoint at 6 mM NaCl). In 1 mM phosphate buffer, the stability of Z-poly(dG-dC)dien-Pt(0.12) is greatly affected by the presence of small amounts of EDTA. Poly(dG-dC) . poly(dG-dC) modified by cis-Pt and trans-Pt complexes do not adopt the Z-form even in high salt concentration.  相似文献   

9.
10.
Poly(dG-dC).poly(dG-dC) was modified by the reaction with 4-hydroxyaminoquinoline 1-oxide (4HAQO) in the presence of seryl-AMP. The conformations of 4HAQO-modified poly(dG-dC).poly(dG-dC) and of poly(dG-dC).poly(dG-dC) were studied by circular dichroism spectra under various salt concentration conditions. 4HAQO residues to guanine bases are inefficient in inducing the transition of poly(dG-dC).poly(dG-dC) from B-form to Z-form conformation. We have elicited monoclonal antibodies against 4HAQO-poly(dG-dC).poly(dG-dC). They were characterized using enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and binding to supercoiled DNA. These antibodies reacted with 4HAQO-poly(dG-dC).poly(dG-dC) specifically but not with 4HAQO-modified DNA or poly(dG).poly(dC). However, they cross-reacted with N-acetoxy-2-acetylaminofluorene-modified poly(dG-dC).poly(dG-dC) in Z-form conformation. These monoclonal antibodies may recognize a unique conformation in poly(dG-dC).poly(dG-dC) after 4HAQO modification.  相似文献   

11.
The effect of 3-aminobenzamide, a potent inhibitor of poly(ADP-ribosyl)ation, on UV-induced DNA excision repair was investigated. HeLa cells were treated with DNA replication inhibitors, hydroxyurea (HU) and 1-beta-D-arabinofuranosyl cytosine (araCyt), before and after ultraviolet light (UV) irradiation, to accumulate DNA single-strand breaks. The activity of poly(ADP-ribosyl)ation measured in the permeable cell system of HeLa cells was enhanced in a UV dose-dependent manner after the combined treatment with HU and araCyt in vivo. However, DNA repair synthesis in vitro was not affected by addition of 1 mM 3-aminobenzamide or nicotinamide, while incorporation of [3H]NAD in the same system was completely inhibited. Furthermore, neither the magnitude of UV-induced DNA single-strand breaks accumulated by the combined treatment of HU and araCyt nor the rate of their rejoining after release from the HU and araCyt block were influenced even in the presence of 10 mM 3-aminobenzamide. As the cytotoxicity of UV irradiation was significantly potentiated by 5 mM 3-aminobenzamide, these results suggest that poly(ADP-ribosyl)ation is involved in a process other than DNA excision repair induced by UV irradiation.  相似文献   

12.
Modifications of circular DNA by photoalkylation   总被引:2,自引:0,他引:2  
The effects of photoalkylation on superhelical PM2 DNA were examined. The chief product was 8-(2-hydroxy-2-propyl)guanine, formed exclusively in sequences of alternating purines and pyrimidines. Other purine damages included 8-(2-hydroxy-2-propyl)adenine and smaller quantities of two uncharacterized adenine products. DNA strand breaks were formed with increasing irradiation. A small quantity of thymine-containing photodimers was formed. Photoalkylation of poly(dG-dC):poly(dG-dC) reduced the concentration of salt required to effect inversion of the circular dichroic spectrum. This suggests that photoalkylation induces the transition of poly(dG-dC):poly(dG-dC) from the right-handed B form of DNA to the left-handed Z form.  相似文献   

13.
Escherichia coli endonuclease III and mammalian repair enzymes cleave UV-irradiated DNA at AP sites formed by the removal of cytosine photoproducts by the DNA glycosylase activity of these enzymes. Poly(dG-[3H]dC) was UV irradiated and incubated with purified endonuclease III. 3H-Containing material was released in a fashion consistent with Michaelis-Menten kinetics. This 3H material was determined to be cytosine by chromatography in two independent systems and microderivatization. 3H-Containing material was not released from nonirradiated copolymer. When poly(dA-[3H]dU) was UV irradiated, endonuclease III released 3H-containing material that coeluted with uracil hydrate (6-hydroxy-5,6-dihydrouracil). Similar results are obtained by using extracts of HeLa cells. There results indicate that the modified cytosine residue recognized by endonuclease III and the mammalian enzyme is cytosine hydrate (6-hydroxy-5,6-dihydrocytosine). Once released from DNA through DNA-glycosylase action, the compound eliminates water, reverting to cytosine. This is consistent with the known instability of cytosine hydrate. The repairability of cytosine hydrate in DNA suggests that it is stable in DNA and potentially genotoxic.  相似文献   

14.
Infrared spectroscopy was used to study the structures and transitions in hydrated gels of double-helical poly(dG-dC) complexed with the metal carcinogens Cd(II) and Ag(I). For one Cd(II) per ten nucleotides (r = 0.1), the B structure was stable at high and moderate hydrations with D2O and the B and Z structures coexisted at low hydrations. For poly(dG-dC) with Cd(II) at r = 0.2 to 0.35, the Z structure was stable at high hydrations (94% r.h. for r = 0.2). At a given value of hydration, H2O gave a higher content of Z structure than D2O. Cd(II) most likely binds to guanine residues at N7 in both the B and Z forms of poly(dG-dC) but binding to guanine N3 can not be excluded. It is unlikely that Cd(II) binds to cytosine residues at the r values studied and the cytosine residues did not protonate at N3 as Cd(II) bound to guanine residues. Poly(dG-dC) with Ag(I) at r = 0.2 to 0.36, existed in a B-family structure which is different from the B-family structure of the type I complex of Ag(I) and calf-thymus DNA. Poly(dG-dC) with Ag(I) did not assume the Z structure at lower hydrations even though NO3- was present in the sample. Ag(I) differs from other soft-metal acids which promote the Z structure. Ag(I) most likely binds to the guanine N7 or N3 and not to cytosine residues. Cytosine residues did not protonate at N3 as Ag(I) was bound to guanine.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

15.
Two derivatives of the alpha/beta-type small acid-soluble spore protein (SASP) SspCwt have been constructed, each containing a residue potentially useful for physico-chemical analysis of protein-protein or protein-DNA interactions. In one mutant protein (SspCtrp) residue 27 (Met) was replaced by Trp; in the second (SspCcys) residue 48 (Asn) was replaced by Cys. Both mutant proteins were expressed in Bacillus subtilis spores at levels similar to those of SspCwt, and SspCcys and SspCtrp restored ultraviolet light (UV) resistance and plasmid negative supercoiling in spores lacking major alpha/beta-type SASP to levels similar to those restored by SspCwt. While the purified mutant proteins bound more weakly to DNA than SspCwt, all three had the same relative affinity for different DNAs, ie poly(dG).poly(dC) greater than poly(dG-dC).poly(dG-dC) greater than pUC19, and purified SspCcys and SspCtrp gave the same pattern of DNase protected bands with pUC19 as SspCwt. Binding of SspCcys or SspCtrp to poly(dG).poly(dC) in vitro also prevented the formation of cyclobutane type cytosine dimers upon UV irradiation, as does binding of SspCwt. These data indicate that the two mutant proteins are extremely similar to SspCwt in their interaction with DNA, and thus may be useful in probing SASP-SASP and SASP-DNA interactions directly by physical or chemical techniques. Indeed, binding of SspCtrp to poly(dG).poly(dC) resulted in a 2.5-fold enhancement of the proteins Trp fluorescence.  相似文献   

16.
A Z-DNA binding protein isolated from D. radiodurans   总被引:2,自引:0,他引:2  
A DNA binding protein isolated from D. radiodurans changes CD-spectrum of Z-form poly(dG-dC) X poly(dG-dC). We have found that a positive band at 268 nm is converted close to that of B-form in the presence of the protein. Concomitantly, a negative band at 295 nm shown by Z-form poly(dG-dC) X poly (dG-dC) was weakened by the protein but not by albumin. Such changes in the CD-spectra were not induced by the protein and by albumin when they were mixed with Z- or B-form poly(dG-me5dC) X poly(dG-me5dC) or with B-form poly(dG-dC) X poly(dG-dC). The protein formed a complex preferentially with Z-form poly(dG-dC) X poly(dG-dC).  相似文献   

17.
The reactions of poly(dG-dC).poly(dG-dC) and (dG-dC)10 insert in the plasmid pGC20 with N-methyl-bis(2-chloroethyl)-amine (nitrogen mustard, HN-2) have been studied. It is shown that nitrogen mustard does not induce the B----Z transition in poly(dG-dC).poly(dG-dC), but produces fixation of the polynucleotide Z-conformation once this exists. In the case of pGC20 plasmid DNA, nitrogen mustard also fixes Z-form of the (dG-dC)-insert. The rate constant of the reaction of nitrogen mustard with guanine in the polynucleotide (k = 9,0.10(-3) min-1) is about one-third of that for the fixation of Z-form of the (dG-dC)-insert in the plasmid (k1 = 2,8.10(-2) min-1) which is attributed to a greater rate of formation of diguanyl derivative in the opposite DNA chains. It is suggested that nitrogen mustard is capable of fixing the Z-form DNA not only in vitro, but also in vivo.  相似文献   

18.
Mouse monoclonal antibody was elicited with 4-nitroquinoline 1-oxide (4NQO) modified poly(dG-dC).poly(dG-dC) and was characterized using enzyme-linked immunosorbent assay and radioimmunoassay. The antibody reacted specifically for 4NQO-poly(dG-dC).poly(dG-dC) but not for 4NQO modified DNA and synthetic polynucleotides such as poly(dG).poly(dC). The antibody crossreacted slightly with brominated or N-acetoxy-2-acetylaminofluorene modified poly(dG-dC).poly(dG-dC) known to adopt Z-conformation. The antibody may recognize unique conformational change in poly(dG-dC).poly(dG-dC) modified by 4NQO. The antibody should be useful for the detection of conformational change in DNA induced by chemical carcinogens.  相似文献   

19.
The sensitivity of S1 nuclease to cis- and trans-(NH3)2PtCl2 modified DNAs is examined as a function of the level of cis- and trans-(NH3)2PtCl2 bound, the % (G+C) content in DNA from different sources and the sequence dependence in poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC). The extent of DNA digested increases with increasing levels of either isomer and is inversely influenced by the % (G+C) content of the DNA. However, the difference in the extent of digestion between the cis-and trans-(NH3)2PtCl2 modified DNAs at equivalent levels of bound isomer follows the order, calf-thymus greater than M. lysodeikticus greater than poly(dG-dC).poly(dG-dC). While there is virtually no difference in the digestion profiles for poly(dG-dC).poly(dG-dC) modified with the two isomers, there is a striking difference in the extent of digestion between cis- and trans-(NH3)2PtCl2 modified poly(dG).poly(dC). These results are discussed in light of the possible modes of binding for cis-(NH3)2PtCl2, previously reported findings on modified DNA and possible implications for modifications in cellular chromatin.  相似文献   

20.
B Jollès  L Chinsky  A Laigle 《Biochimie》1984,66(2):101-104
Resonance Raman Spectroscopy allows a selective study of the bases of DNA and therefore of the interactions of these bases with ligands. This technique is also sensitive to structural modifications. We show here that, first, the structures of native poly(dA-dT).poly(dA-dT) and poly(dA).poly(dT) are not the same and that, secondly, it is possible to characterize the B----Z transition of poly(dG-dC).poly(dG-dC). The study of the Raman hypochromism during the thermal denaturation of the polynucleotides reveals that the stacking of the adenines in poly(dA).poly(dT) is near that observed in poly(rA) but differs of this stacking in poly(dA-dT).poly(dA-dT). The enhancement of the intensity of the guanine line at 1193 cm-1 and of the cytosine lines at 780 cm-1, 1 242 cm-1 and 1268 cm-1 as well as the shift of the guanine line at low frequency should allow to characterize a small proportion of base pairs in Z form in any DNA.  相似文献   

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