Rotational diffusion of the 7-diethylamino-4-methylcoumarin C1 dye molecule in polar protic and aprotic solvents.

Fluorescence anisotropy decays of the 7-diethylamino-4-methylcoumarin C1 in various polar solvents of different viscosities and hydrogen bond donor/acceptor character have been recorded by means of the fluorescence upconversion and time-correlated single photon counting techniques. The resulting characteristic times for the rotational diffusion fall into two classes with regards to the viscosity-dependency: n-alcohols and "other" solvents. This deviation from the simple Stokes-Einstein-Debye model may be interpreted in terms of rotation of the coumarin molecule under two different hydrodynamic boundary-conditions ("stick" or "slip") in the two solvent classes. Possible explanations for this behaviour are discussed, and in particular solvent attachment and additional dielectric friction. Both these phenomena may in fact, under certain conditions, explain our findings. Our opinion, however, is that the dielectric friction model offers a more realistic picture of the additional rotational friction experienced by C1 in n-alcohols.

Solvent-dependent C-OH homolysis and heterolysis in electronically excited 9-fluorenol: the life and solvation time of the 9-fluorenyl cation in water.

The primary pathways of the photodecomposition of 9-fluorenol (FOH) were studied in polar and nonpolar solvents by use of laser flash-photolysis with a resolution time of 10 ps. In solvents of high polarity, that is, in 1,1.1,3,3,3-hexafluoroisopropanol (HFIP), 2,2,2-trifluoroethanol (TFE), formamide or water, the fluorenyl cation, F+, forms by heterolytic C-O bond cleavage. In H2O, the initial (10 ps) spectrum of F+ has lambdamax at <460 nm. This absorption red-shifts with T = 25 ps to the "classical" spectrum with lambdamax = 510-515 nm. This process is assigned to the solvation of the initial "naked" cation, or rather, the contact ion pair. The lifetime of the solvated fluorenyl cation in H2O (or D2O) and TFE was measured to be tau 20 ps and 1 ns, respectively. In solvents of lower polarity such as alkanes, ethers and alcohols, the long-lived (tau 1/2 1 micros) fluorenyl radical, F., (lambdamax = 500 nm) forms through homolytic C-O cleavage. In addition to the radical and the cation, the vibrationally relaxed excited singlet state of FOH is seen with its absorption at approximately 640 nm; its lifetime is strongly dependent on the solvent, from 10 ps for formamide to 1.7 ns for cyclohexane. The rate constant for singlet decay increases exponentially with the polarity of the solvent (as expressed by the Dimroth-Reichardt ET value) or with the Gutmann solvent acceptor number. The relaxation of S1 to S0 is accompanied by homolytic C9-O bond cleavage (except in HFIP, TFE, and water, where S1 is not seen).

Depopulation of highly excited singlet states of DNA model compounds: quantum yields of 193 and 245 nm photoproducts of pyrimidine monomers and dinucleoside monophosphates.

Formation of uracil and orotic acid photodimers, uridine and 5'-UMP photohydrates, TpT photodimers and (6-4)photoproducts, dCpT photohydrates and (6-4)photoproducts and UpU, CpC and CpU photohydrates were studied in neutral deoxygenated aqueous solution at room temperature upon irradiation at either 193 or 254 nm. The photoproducts were identified and quantified and the contribution from photoionization to substrate decomposition, using lambda irr = 193 nm, was separated. The ratio of the quantum yields of respective stable products, eta = phi 193/phi 254, is indicative of the yield of internal conversion from the second to the first excited singlet state, S2-->S1. For the observed photodimers eta decreases from 0.94 for uracil to 0.7 for TpT and further to 0.55 for orotic acid. For the (6-4)photoproducts of TpT and dCpT eta = 0.5-0.8 and for the photohydrates in the cases of UpU, CpC, CpU and dCpT eta ranges from 0.55 to 1.

Ultraviolet (193, 216 and 254 nm) photoinactivation of Escherichia coli strains with different repair deficiencies.

Escherichia coli K12 bacteria strains AB1157 (repair-deficient wild-type, uvrA+ recA+), AB1886 (urvA-), AB2463 (recA-) and AB2480 (urvA- recA-) were exposed to 254 nm germicidal UV and 216 or 193 nm laser radiation. The mean lethal incident dose (D37) for AB1157 does not change significantly with wavelength, whereas it increases for the other strains on going from lambda irr = 254 to 193 nm, e.g. eightfold for AB2480. Quantum yields for inactivation, due to light absorbed by the chromosomal DNA, have been estimated from the D37 values. The large differences in D37 between uvrA+ and uvrA- strains indicate a significant contribution of pyrimidine dimers and (6-4) photoproducts to photodamage of DNA in the cells. The formation of dimers even with lambda irr = 193 nm is supported by the result that the photoreactivable sector is larger than 63%. Inactivation of E. coli upon irradiation at 193 and 216 nm is therefore due to damage to intracellular DNA rather than to membrane or protein damage as is the case for mammalian cells. The ratio of the lethal doses for AB1157 vs AB2480 is approximately 900 with lambda irr = 254 nm, but only 160 with lambda irr = 193 nm. We propose that this is due to the better repair of photodimers and (6-4) photoproducts than of damage induced by photoionization via upper excited states. Irradiation at 193 nm inactivates AB1157 mainly by damage due to photoionization and AB2480 by damage due to photodimers in the chromosomal DNA.

Damage to uracil- and adenine-containing bases, nucleosides, nucleotides and polynucleotides: quantum yields on irradiation at 193 and 254 nm.

Photoreactions, such as base release and decomposition of the base moiety, induced by either 20 ns laser pulses at 193 nm or continuous 254 nm irradiation, were studied for a series of uracil and adenine derivatives in neutral aqueous solution. The quantum yield of chromophore loss (phi cl) depends significantly on the nature of the nucleic acid constituent and the saturating gas (Ar, N2O or O2). In the case of polynucleotides the destruction of nucleotides was measured by high-performance liquid chromatography after hydrolysis; the quantum yields (phi dn) are comparable to those of chromophore loss or larger. The phi cl and phi dn of 0.04-0.1 for poly(U) and poly(dU), obtained for both wavelengths of irradiation, are due to processes originating from the lowest excited singlet state, i.e. formation of photohydrates and photodimers, and a second part from photoionization using lambda irr = 193 nm. Irradiation at 193 nm effectively splits pyrimidine dimers and thus reverts them into monomers. The quantum yield for release of undamaged bases (phi br) from nucleosides, nucleotides and polynucleotides upon irradiation at 254 nm is typically phi br = (0.1-1) x 10(-4). Breakage of the N-glycosidic bond is significantly more efficient for lambda irr = 193 nm, e.g. phi br = 1.1 x 10(-3), 0.8 x 10(-3), 4.3 x 10(-3) and 0.5 x 10(-3) for poly(A), poly(dA), poly(U) and poly(dU) in Ar-saturated solution, respectively. Enhanced phi values for lambda irr = 193 nm, essentially for adenine and its derivatives, are caused by photoprocesses that are initiated by photoionization.

Are enzymatically produced single-strand breaks involved in UV-induced inactivation of plasmid DNA?

pBR322 plasmid DNA was exposed to 254 nm UV radiation and examined for enzymatically produced single-strand break (sbb) and double-strand break (dsb) formation by treatment with an extract containing the proteins of Escherichia coli (AB1157 (uvrA+ recA+) and AB2480 (uvrA- recA-)). Enzymatic conversion of the 254 nm-induced lesions into ssbs on treatment with an extract from AB1157 was observed, but not conversion into dsbs. The rate of enzymatic ssb formation in the AB1157 extract is initially higher than in the AB2480 extract, the sbb formation levels off leading to plateau values with increasing incubation time. The rate of ssb formation in the AB2480 extract is initially lower, but does not level off, and the ssb yield becomes larger at longer incubation times than that with the AB1157 extract. The biological inactivation of the plasmids was measured as a function of 254 nm fluence by transformation of E. coli AB1157 and AB2480. Inactivation with AB2480 is mainly due to a single photoproduct, a cyclobutane-type pyrimidine dimer, per DNA molecule. Inactivation with AB1157 occurs with a quantum yield which is virtually identical with that of the plateau values of enzymatic ssb formation, as measured by incubation in the AB1157 extract. A possible interpretation is that the formation of irreparable ssbs is the lethal step in the sequence of events leading to inactivation of plasmid DNA in the repair wild-type strain. The quantum yield of inactivation is 10-20 times smaller for transformation of AB1157 than for AB2480, indicating that enzymatic repair of photolesions of the plasmid occurs in AB1157.

Photolesions and biological inactivation of plasmid DNA on 254 nm irradiation and comparison with 193 nm laser irradiation.

Plasmid pTZ18R and calf thymus DNA in aerated neutral aqueous solution were irradiated by continuous 254 nm light. The quantum yields are phi ssb = 4.0 x 10(-5) and phi dsb = 1.4 x 10(-6) for single- and double-strand break formation, respectively, phi br = 2.3 x 10(-5) for base release, phi dn = 2.1 x 10(-3) for destruction of nucleotides, and phi icl approximately phi lds approximately 1 x 10(-6) for interstrand cross-links and locally denatured sites, respectively. The presence of Tris-HCl/ethylenediaminetetraacetic acid (10:1, pH 7.5) buffer strongly reduces phi ssb. The corresponding phi values, obtained on employing pulsed 193 nm laser irradiation, are much larger than those using lambda irr = 254 nm. This is ascribed to a contribution of chemical reactions induced by photoionization, which is absent for 254 nm irradiation. The quantum yields of inactivation of plasmid DNA (lambda irr = 254 nm) were measured by transformation of the Escherichia coli strains AB1157 (wild type), phi ina (1157) = 1.6 x 10(-4), AB1886 (uvr-), phi ina (1886) = 4.2 x 10(-4), AB2463 (rec-), phi ina (2463) = 4.1 x 10(-4) and AB2480 (uvr- rec-), phi ina (2480) = 3.1 x 10(-3). The quantum yields of inactivation of plasmid DNA are compared with those of the four E. coli strains (denoted as chromosomal DNA inactivation) obtained from the literature. The results for E. coli strain AB2480 show that the chromosomal DNA and the plasmid DNA are both inactivated by a single pyrimidine photodimer per genome.(ABSTRACT TRUNCATED AT 250 WORDS)

Photolesions in DNA upon 193 nm excitation.

Aqueous solutions of plasmid (pBR322 and pTZ18R) and calf thymus DNA were excited by 20 ns laser pulses at 193 nm. The quantum yields of single-and double-strand break formation, interstrand cross-links, locally denatured sites, (6-4)photoproducts and biological inactivation (phi ssb, phi dsb, phi icl, phi lds, phi 6-4 and phi ina, respectively) were measured. The quantum yields are virtually independent of intensity, demonstrating a one-quantum process. The obtained values in aerated neutral solution in the absence of additives are phi ssb approximately 1.5 x 10(-3), phi dsb approximately 0.06 x 10(-3) (dose: 10-200 J m-2), phi icl approximately phi lds approximately 0.1 x 10(-3) and phi 6-4 = 0.5 x 10(-3). Both phi ssb and phi dsb decrease strongly with increasing concentrations of TE buffer (0.01-10 mM). Biological inactivation of the pTZ18R plasmid was determined from the transformation efficiency of Escherichia coli bacteria strains AB1157, AB1886 uvr and AB2480 uvr rec; the phi ina values are 1.4 x 10(-3), 2.1 x 10(-3) and 3 x 10(-3), respectively. The monoexponential survival curves in all cases show that a single damage site leads to inactivation (one single hit). The biological consequences of different photoproducts are discussed.

Base release from DNA and polynucleotides upon 193 nm laser excitation.

Release of bases form calf thymus DNA and three polynucleotides, induced by 20 ns excitation at 193 nm in aqueous solution at pH 7, was detected by HPLC. The quantum yields of formation of free bases (phi B) from double-stranded DNA (0.4 mM) are independent of intensity, indicating a one-quantum mechanism of N-glycosidic bond cleavage. The phi B values increase in the order guanine, thymine, adenine, cytosine, the latter being phi C approximately 7 x 10(-4) for double-stranded DNA under Ar and O2. The larger phi B values in N2O-saturated solution, e.g., phi C = 1.2 x 10(-3), are ascribed to additional base release via OH-adduct radicals. The phi B values of homopolynucleotides increase in the order poly(G), poly(A) and poly(C), e.g. phi C = 7 x 10(-3) under Ar, as do the efficiencies for base release per radical cation (eta B). A comparison of the eta B values with the efficiencies of single-strand breakage for poly(C), poly(A) and DNA shows a similar trend; both are markedly larger for pyrimidines than for purines. Pathways to undamaged bases, initiated from base radical cations, are proposed.

Two-quantum photoprocesses in DNA under picosecond laser UV irradiation at 216 and 270 nm.

It is demonstrated that at high power picosecond laser irradiation of 216 and 270 nm, two-quantum photodestructions of the DNA secondary structure, such as interstrand covalent crosslinks, "weak" crosslinks, and B----C conformational transition, take place. Thermal effects do not contribute to the observed effects.