Thymine-dimer and action-spectrum evidence for indirect photoreactivation in Escherichia coli

Abstract— Cells of Escherichia coli B and B phr- were labeled with tritiated thymidine, exposed to inactivating ultraviolet radiation at 254 nm, given photoreactivation (PR) treatment immediately thereafter, and then immediately hydrolyzed and assayed for thymine-containing dimers. It was found that (1) PR treatment of strain B phr- does not split thymine dimers and (2) the amount of splitting of thymine dimers in strain B at 334 nm is only 45 per cent of the amount of splitting observed at 405 nm for the same amount of biological PR. These findings show that all of the PR in E. coli B phr-, and part of the PR at 334 nm in E. coli B, is indirect (does not use PR enzyme) and is not due to thymine-dimer splitting. Action spectra for PR in Escherichia' coli strains B_s-1 and B/r were obtained. At wavelengths below 366 nm (the indirect PR region), PR is relatively much more efficient in strain B/r in logarithmic phase than in strain B/r in stationary phase or in strain B_s-l. This is consistent with the expectation that indirect PR would not be exhibited by strains, such as B_s-1 that lack dark-repair ability, or by certain strains in the stationary phase, such as B/r, which, upon plating, have a ‘built-in’ growth delay that can permit optimal dark repair, but indirect PR would be exhibited by intermediate cases, such as in strain B (which is less resistant to u.v. than strain B/r) or in strain B/r in logarithmic phase. These findings support the hypothesis that indirect PR results from enhancement of dark repair.     

Liquid-holding effects in U.V.-irradiated phage T3

Abstract— Holding complexes of u.v.-irradiated (254 nm) T3 phage in E. coli B/r cells for several hours at 37°C in buffer, or broth with chloramphenicol, affects the phage survival in at least two different ways: (1) by enhancing excision repair, resulting under certain conditions in liquid-holding recovery (LHR), and (2) by destroying the phage (holding inactivation). LHR is most apparent in buffer containing 20 μg ml^-1 chloramphenicol (CAP). It is expressed by as much as a 10–fold increase in the fraction of complexes that display host-cell reactivation (resulting from excision repair), but the percentage of u.v. lesions repaired within repair-proficient complexes is slightly decreased. LHR is not observed if T3 infects the repair-deficient strain B_s-1. Holding inactivation is readily observed with unirradiated phage complexes in broth containing CAP. The response of irradiated-phage complexes to liquid-holding conditions is more complex: holding inactivation is less effective for irradiated than for unirradiated phage DNA (i.e. the irradiated DNA is to some extent ‘protected’), and processes leading to LHR are superimposed. Thus under certain holding conditions one observes the paradoxical phenomenon that the viable titer of irradiated phage is several times higher than that of unirradiated phage. The nature of holding inactivation is not known, nor is the mechanism by which irradiated DNA is partially protected against it. Holding inactivation does not require protein synthesis; it is rather enhanced at high CAP concentration and seems to be favored by otherwise active cell metabolism. At high CAP concentrations (200–400 μg ml^-1, as compared to 20 μg ml^-1) irradiated-phage complexes show neither LHR nor protection against holding inactivation. Likewise they fail to undergo some step by which the phage DNA becomes insensitive to repair inhibition by caffeine.     

Induction of single-strand breaks (alkali-labile bonds) in bacterial and phage DNA by near UV (365 nm) radiation

Abstract —Irradiation at 365 nm results in the induction of approximately 2–4 times 10^-6 and 1-2times 10^-6 single-strand breaks (alkali-labile bonds) per 10⁸ daltons per J m^-2 in extracted phage T4 DNA and in Escherichia coli bacterial DNA, respectively. The rate of break induction in DNA of intact phage is approximately one-fourth that for extracted phage DNA. 2-aminoethylisothiouronium bromide-HBr protects against break induction in both phage systems. No breaks are induced in the DNA of bacteria irradiated under anaerobic conditions over the dose range tested. Possible induction mechanisms are suggested. Consideration is given to the relative importance of pyrimidine dimers and single-strand breaks in the bactericidal action of 365 nm radiation.     

PHOTOSENSITIZATION OF SUPERCOILED DNA DAMAGE BY 5,6-DIHYDROXYINDOLE-2-CARBOXYLIC ACID, A PRECURSOR OF EUMELANIN

The photosensitizing or photoprotecting action of 5,6-dihydroxyindole-2-carboxylic acid (DICA), an intermediate in the biosynthesis of eumelanins, was investigated. Under irradiation at 313 nm, aqueous buffered solutions of DICA (22.5 μW) photosensitized the cleavage of phage φX174 DNA. The number of single strand breaks (SSB) depended on the dose of irradiation and was more important in the absence than in the presence of oxygen. In the presence of oxygen, the quantum yield of SSB was around 6′10 ⁷ (Φ_SSB) The influence of specific scavengers, such as mannitol, sodium azide or superoxide dismutase, indicated that hydroxyl radicals, superoxide anions and perhaps singlet oxygen were involved in these processes. The increase in SSB in D₂O was also indicative of the participation of singlet oxygen. Comparative experiments performed with indole-2-carboxylic acid (IC), a dehydrox-ylated analog of DICA, showed that this compound, although lacking a phenol group, also photosensitized DNA cleavage via a mechanism involving hydroxyl radicals. Various sources of these radicals were envisioned. Furthermore, under our conditions, DICA was not found to photoinduce the formation of DNA dimers: No increase in SSB was observed in DNA irradiated in the presence of DICA, after treatment by phage T4 endonuclease V (an enzyme that selectively cuts DNA at dimer sites), whereas, in contrast, a significant increase in SSB was detected after treatment of DNA irradiated alone. So it appears that DICA may both photosensitize DNA cleavage and reduce UV-induced DNA dimer formation.     

Correlation of photoreactivation of ultraviolet-induced killing with dimer splitting before and after DNA replication in an excisionless strain of Escherichia coli

Abstract— Splitting of thymine-containing dimers was compared quantitatively with photoreactivation (PR) of killing induced by ultraviolet radiation (254 nm) in a uvrA (excisionless) strain of E. coli. Immediately after irradiation, the splitting rate (number of dimers split/genome/unit PR dose) agreed well with the PR rate of the cells (rate of recovery from photoreactivable lethal damage converted into an ‘estimated’ number of dimers split/genome/unit PR dose). After 4 h of incubation of cells in nutrient medium, the maximal fraction of splittable dimers decreased, as did the maximal fraction of photoreactivable lethal damage. However, the initial splitting rate after incubation was equal to that before incubation. During the 4-h incubation, the heavily irradiated uvrA cells did not divide but became filamentous and their DNA increased about 70 per cent. It is concluded that roughly half of the dimers in DNA that has replicated after ultraviolet irradiation are split as efficiently as those in DNA that has not replicated.     

Photoreactivation of killing in Streptomyces. II. Kinetics of formation and photolysis of pyrimidine dimers and adducts in S. coelicolor and S. griseus PHR-1

Abstract— A pyrimidine adduct, 6-4‘-[pyrimidine-2’-one] thymine (PO-T)?, observed in DNA hydrolysates of 254-nm ultraviolet (u.v.) irradiated conidia of Streptomyces coelicolor, increases linearly with u.v. dose up to 2 × 10⁵ ergs/mm². Yields of thymine dimer (T○) and uracil-thymine dimer (U○) level off at much lower doses. Initial relative rates of formation of these u.v. photoproducts are: 1:1.3:4.8 for PO-T, T○ and U○, respectively. Similar results were obtained with a Streptomyces griseus mutant, PHR-1. An equation is derived to estimate the ratio of the amount of PO-T to the total amount of thymine-derived photoproducts at low (biological) u.v. doses. The observed PO-T fractions compare well with the calculated values. Rapid photolysis of the precursor of PO-T was observed by post-u. v. treatment at 313 nm of conidia of S. coelicolor and of S. griseus PHR-1. The photolysis was much slower at 365 nm and did not occur at all at 405 nm. Pyrimidine dimers were not appreciably affected by post-u. v. treatment at the above wavelengths in these Streptomyces strains. Both of these strains are phenotypically photoreactivation-deficient, and the present results indicate that they do not possess active photoreactivating enzyme. In earlier papers[3,4,5], the pyrimidine adduct found in acid hydrolysates of DNA was loosely referred to as “uracil-thymine adduct (U-T adduct)”. Such terminology is not strictly correct. The pyrimidine adduct in acid hydrolysates is PO-T (sometimes called P₂B), which could theoretically result from removal of ammonia from a C-T adduct or removal of water from a U-T adduct (see [6]).     

REPAIR OF NEAR (365 nm)- AND FAR (254 nm)-UV DAMAGE TO BACTERIOPHAGE OF ESCHERICHIA COLI

Abstract: Intact bacteriophage have been irradiated at 365 nm or at 254 nm and then analysed for DNA photoproducts or injected into their bacterial host to test susceptibility of the damage to both phage and host-cell mediated repair systems. Both thymine dimers and single-strand breaks are induced in the phage DNA by 365 nm radiation. The dimers appear to be the major lethal lesion (approximately 2 dimers per lethal event) in both repair deficient bacteriophage T4 and bacteriophage λ. after irradiation with either 254 nm or 365 nm radiation. Damage induced in T4 by either wavelength is equally susceptible to x -gene reactivation (repair sector approximately 0.5). v -gene reactivation acts on a larger fraction of the near-UV damage (repair sector of 0.82 at 365 nm as against 0.66 at 254 nm). The host-cell mediated photoreactivation system is only slightly less effective for near-UV damage but host-cell reactivation (as measured by comparing survival of phage λ. on a uvr⁺ and a uvr^- host) is effective against a far smaller sector of near-UV damage (0.35) than far-UV damage (0.85). Weigle-reactivation (far-UV induced) of near-UV damage to phage λ is not observed. The results suggest that unless the near-UV damaged phage DNA is repaired immediately after injection. the lesions rapidly lose their susceptibility to repair with a consequent loss of activity of the phage particles.     

Absence of repair replication following ultraviolet irradiation of an excision-deficient mutant of Escherichia coli

The excision -repair of damaged DNA in bacteria and other systems probably requires at least three enzymes to carry out the following steps in sequence: (1) Recognition of a structural distortion in the DNA and the production of an endonucleolytic cleavage of the damaged strand near the lesion. (2) The simultaneous peeling back of the damaged strand and resynthesis of the excised region, with eventual cleavage of the damaged segment from the DNA. (3) The rejoining of the newly synthesized strand to contiguous parental DNA. Evidence for all three steps has been obtained from in vivo studies. The E. coli DNA polymerase has been shown to carry out step # 2 in vitro [1] and the polynucleotide ligase has the required specificity for step # 3[2–4]. An enzyme responsible for step # 1 has been purified from Micrococcus lysodeikticus [5,6] but not from E. coli, although a class of u.v. sensitive mutants in E. coli has been shown to be defective in this step in the repair sequence. In such mutants the release of pyrimidine dimers from the damaged DNA is not observed during post-irradiation growth of u.v. irradiated cultures [7]. It would be predicted, as a consequence, that the next step, non-conservative repair replication, would not be seen in these mutants. Hanawalt and Petti-john showed this to be true for the double mutant E. coli B_8-1 that includes a deficiency in dimer excision [8]. In the present study we have looked more closely at an E. coli K-12 strain that has only the uvrA6 deficiency that results in inability to excise pyrimidine dimers.     

Crystal engineering with heteroboranes. I. 1-Carboxy-1,2-dicarba-closo-dodecaborane(11)

The title compound, C₃H₁₂B₁₀O₂ or 1-COOH-1,2-closo-C₂B₁₀H₁₁, forms centrosymmetric dimers through intermolecular hydrogen bonding between the carboxylic acid groups, resulting in the formation of an eight-membered ring [R(8)]. The C=O bond of the carboxylic acid group almost eclipses the unsubstituted cage C atom, with a C—C—C—O torsion angle of 2.6 (2)°.     

新型三齿多吡啶钴(II)、钌(II)配合物的合成、表征及其与DNA的作用研究

合成了两种新型三齿多吡啶钴(II)和钌(II)的混配配合物[Co(TolylTPy)(H₂Bzimpy)]Cl₂ [TolylTPy＝4'-对甲基苯 基-2,2':6',2'-三联吡啶, H₂Bzimpy＝2,6-二(苯并咪唑-2)吡啶] (A)和Ru(TolylTPy)(Bzimpy) (B). 用元素分析, IR, ¹H NMR等对它们进行了表征, 测定了配合物B的晶体结构, 用电子吸收光谱、荧光光谱等研究了配合物与小牛胸腺DNA(CTDNA)的相互作用及其对pBR322 DNA的断裂作用. 结果表明, 配合物A和B与CTDNA的作用属静电结合, 凝胶电泳实验说明配合物A在310 nm光辐射15 min, 可使超螺旋pBR322 DNA断裂为开环缺口型和线型DNA.     

The electronic structures and spectra of proflavine,acridine orange,and their DNA complexes

The electronic structure of the proflavine cation is studied by the SCF –ASMO –CI method using the Pariser–Parr–Pople approximations. It is shown that the band at 445 mμ may be assigned to the ¹A₁ → ¹B₁, transition polarized along the long axis of the molecule. The bands in the neighbourhood of 260 mμ, which are composed of three absorption bands, are tentatively assigned to the ¹A₁ → ¹B₁, ¹A₁ → ¹B₁, and ¹A₁ → ¹A₁ transitions, respectively, in order of decreasing wavelength. The spectrum of the acridine orange cation may be understood to have the same assignment as that of the proflavine cation. The acridine dye cations are well known for their dimerization with concentration. The intermolecular distances in these dimers are estimated from the band shifts due to the formation of dimers, using the exciton theory. The main contribution to the molecular interaction is shown to be the electrostatic dipole–dipole interaction. Since the first excitation band of the dye molecule which exhibits a remarkable change due to the formation of the DNA–acridine dye complex, is suggested to be polarized along the long axis, preference of the outside stacking or the intercalation model is qualitatively discussed from the spectral shift of the acridine dye bound to the DNA, assuming simple models.     

2‐[2‐(Pyrrolidin‐2‐yl)propan‐2‐yl]‐1H‐pyrrole and its amide derivative 1‐{2‐[2‐(1H‐pyrrol‐2‐yl)propan‐2‐yl]pyrrolidin‐1‐yl}ethanone

In the title compounds, C₁₁H₁₈N₂, (II), and C₁₃H₂₀N₂O, (III), the pyrrolidine rings have twist conformations. Compound (II) crystallizes with two independent molecules (A and B) in the asymmetric unit. The mean planes of the pyrrole and pyrrolidine rings are inclined to one another by 89.99 (11) and 89.35 (10)° in molecules A and B, respectively. In (III), the amide derivative of (II), the same dihedral angle is much smaller, at only 13.42 (10)°. In the crystal structure of (II), the individual molecules are linked via N—H...N hydrogen bonds to form inversion dimers, each with an R²₂(12) graph‐set motif. In the crystal structure of (III), the molecules are linked via N—H...O hydrogen bonds to form inversion dimers with an R²₂(16) graph‐set motif.     

Photochemistry and Photobiology of lmidazole-4(5)-Methylidene Malonic Acid: An Analog of Both E- and Z-Urocanic Acid*

Imidazole-4(5)-methylidene malonic acid (IMMA) may be thought of as having its imidazole and carboxyl functionalities permanently fixed in a configuration that is simultaneously analogous to both E- (trans) and Z- (cis) urocanic acid (UA). Because the UA isomers affect the photoinactivation of bacteriophage single-stranded DNA differently (E-UA increases and Z-UA decreases viral DNA inactivation), IMMA was similarly tested and was found to change the inactivation rate by a factor of 0.43, which is intermediate between the values for E- and Z-UA (1.6 and 0.014, respectively). The IMMA likewise sensitizes double-stranded DNA by a factor of 10.3 compared to a value of 13 for UA. In several ways the effects of IMMA parallel the distinctive effects of UA on the UV inactivation of single- and double-stranded DNA, including the ability to prevent the formation of cyclobutane pyrimidine dimers in irradiated single-stranded DNA and to sensitize a large increase in the formation of those dimers in irradiated double-stranded DNA. The IMMA photodecarboxylates to UA with a low quantum efficiency (ca 1 × 10^?3) and photochemically binds to calf-thymus DNA.     

SENSITIZED PHOTOINACTIVATION OF BACTERIOPHAGE T4

Abstract— The photoinactivation of an osmotic shock resistant mutant of the bacteriophage T 4 can be sensitized by two cationic derivatives of acetophenone. At least part of the sensitized inactivation appears to be due to the formation of thymine dimers in phage DNA by a mechanism which involves triplet excitation transfer from the sensitizer to thymine in the DNA. The photoreactivable sector of the phage inactivated by sensitized irradiation is about 0.6, almost twice as large as the sector for u.v. irradiated phage.     

Photobiology of RNA bacteriophages. II. U.V.-irradiation of f2: effects on extracellular stages of infection and on early replication

Abstract— The effect of u.v. irradiation (2537 Å) on the RNA bacteriophage f2 has been studied with respect to the adsorption of f2 to E. coli K12 (male strain), the penetration of f2-RN A into the host cell and the conversion of the phage nucleic acid to the double-stranded replicative intermediate. The biological parameter most sensitive to u.v. was the plaque-forming ability of the phage. Its loss could be attributed to several factors. (1). A binding of capsid protein to phage nucleic acid interfering with host penetration by the f2-RNA. (2). Desorption of some irradiated phage at 37° from their attachment sites on the host. (3). Molecular alterations in the RNA preventing formation of the replicative intermediate within the host. The relationship of these factors to the lack of photoreactivability of irradiated f2 is discussed.     

Yeast deoxyribodipyrimidine photolyase (photoreactivating enzyme): optimum conditions for activity and competitive inhibition

Abstract— Using flash photolytic techniques and direct chemical measurements of the conversion of the substrate (conversion of thymine dimers in DNA to monomeric thymine), we have determined photolyase concentrations in partially purified preparations of soluble proteins from yeast and have determined under continuous intense light the forward rate constant k₁ for binding of the enzyme and its substrate under a variety of conditions. The ionic requirements and the sharp peak of ionic strength dependence are independent of the species of uni-univalent salts used in the assay. At infinite dilution, the k₁ for denatured DNA, and its ionic strength dependence, both appear identical to the values for native DNA. Both unirradiated denatured and unirradiated native DNA inhibit binding, denatured DNA being 10- to 20-fold more effective. These combined factors have been taken into account to devise a sensitive assay for photoreactivable lesions in unlabeled DNA by competition in a flash photoreactivation reaction. The assay is used to measure dark repair in Micrococcus luteus in complete medium. After a dose of 100 J/m² the wild type of this organism removes photoreactivable lesions (pyrimidine dimers) from its DNA with a half-time of 7 min at 35°C.     

Electronic structure and stability of BP clusters: theoretical calculations for (BP)n (n = 2–4)

The geometry, electronic configurations, harmonic vibrational frequencies, and stability of the structural isomers of boron phosphide clusters have been investigated using density functional theory (DFT). CCSD(T) calculations show that the lowest‐energy structures are cyclic (IIt, IVs) with D_nh symmetry for dimers and trimers. The caged structure for B₄P₄ lie higher in energy than the monocyclic structure with D_2d symmetry (VIs). The B–P bond dominates the structures for many isomers, so that one preferred dissociation channel is loss of the BP monomer. The hybridization and chemical bonding in the different structures are also discussed. Comparisons with boron nitride clusters, the ground state structures of B_nP_n (n = 2, 3) clusters are analogous to those of their corresponding B_nN_n (n = 2, 3) counterparts. © 2005 Wiley Periodicals, Inc. Int J Quantum Chem, 2006     

Novel Enzyme-Linked Aptamer Assay for the Determination of Aflatoxin B1 in Peanuts

Abstract

A novel enzyme-linked aptamer assay is reported for the determination of aflatoxin B₁ (AFB₁). AFB₁ can competitively bind with the immobilized biotin-aptamer and release biotin complementary DNA, leading to the gradual fading of the detection system color with increasing of AFB₁ concentration. In the absence of AFB₁, the biotinylated complementary DNA is not be released from the fixed aptamer. Therefore, the enzyme reaction occurs in the detection system. Under the optimized experimental conditions, the proposed method possessed a wide linear range for AFB₁ from 1 to 80?ng/mL (R² of 0.990) with a low detection limit of 0.36?ng/mL. The method was then applied to detect uncontaminated peanuts fortified with different concentrations of AFB₁. The recovery values were from 82.60% to 94.43%, which indicated the proposed method may be used to detect AFB₁ in food and has potential for the development of test kits.     

Geometry,stability, and isomerization of BnN2 (n = 1−6) isomers

We perform a systematic study on the geometry, stability, nature of bonding, and potential energy surface of low‐lying isomers of planar and cyclic B_nN₂ (n = 1?6) at the CCSD(T)/6‐311+G(d)//B3LYP/6‐311+G(d) level. B_nN₂ (n = 2?4) clusters are structurally similar to pure boron clusters. The evolution of the binding energy per atom, incremental binding energy, and second‐order difference of total energy with the size of B_nN₂ reveals that the lowest energy isomer of B₃N₂ has high stability. B₅N₂ and B₆N₂ possess π‐aromaticity according to Hückel (4n + 2) rule. The aromaticity of some isomers of B₄N₂ and B₆N₂ is examined based on their valence molecular orbitals. At the CCSD(T)/6‐311+G(d)//B3LYP/6‐311+G(d) level, several B₂N₂, B₃N₂, B₄N₂, and B₅N₂ isomers are predicted to be stable both thermodynamically and kinetically, and detectable in future experiments. © 2013 Wiley Periodicals, Inc.     

Action of photoreactivating light on pyrimidine heteroadduct in bacteria

Abstract— In stationary-phase Escherichia coli B/r, photoreactivation (PR) at 313 nm of ultraviolet (u.v.) killing is inefficient compared with PR at 405 nm, and can be explained solely by photoenzymatic reversal of pyrimidine dimers. In Staphylococcus epidermidis, PR shows a maximum at 313 nm, suggesting that this organism shows the Type III PR proposed by Jagger et al.[5] for Streptomyces strains. Reversal of pyrimidine dimers is not sufficient to explain this PR. The mechanism of Type III PR remains unknown. With both S. epidermidis and E. coli B/r, the amount of uracil–thymine heteroadduct in DNA hydrolysates decreases if the cells are given a post-u.v. treatment at 313 nm, but no decrease is observed if the post-u.v. treatment is at 405 nm. The biological significance of this adduct and of its removal is not clear. It may play a role in Type III PR.