PHOTOREPAIR OF NONADJACENT PYRIMIDINE DIMERS BY DNA PHOTOLYASE

Abstract— Photolyases reverse the harmful effects of UV light on cells by converting pyrimidine dimers (Pyr[]Pyr) into two pyrimidine monomers by utilizing near-UV and visible light. Previous work has shown that photolyase repairs T[c,s]T and T[t,s]T in DNA as well as U[]U in RNA, all of which are formed by joining the two adjacent pyrimidines in a light-dependent reaction. In this report, we show that Pyr[]Pyr formed in nonadjacent pyrimidines are also substrates for DNA photolyase.     

9-甲基蒽单体、二聚体和夹心面包对在硅胶上的光物理和光化学性质研究

研究了在硅胶表面上的9-甲基蒽和它的二聚体及其低温光解产物(夹心面包对)的荧光光谱,激发光谱和荧光寿命。发现了一个由二聚9-甲基蒽、三氯甲烷和真空下700℃活化硅胶形成的复合物。该复合物具有与单体和二聚体不同的特征激发光谱和发射光谱,低温下光照容易分解。当分解产物缓缓加热到室温,则复合物几乎完全可以再现。二聚体低温光解产物,夹心面包对,当其缓缓加热到室温,则完全变为单体蒽。     

PHOTOPHYSICS AND PHOTOCHEMISTRY OF PHOTOREACTIVATION

Abstract— Photoreactivating enzyme (PRE) monomerizes cyclobutyl pyrimidine dimers formed in DNA by UV light ( Λ < 300 nm). The enzyme requires near UV and visible wavelengths (300 < Λ < 600 nm) for activity. Possible mechanisms of action of the PRE are suggested by non-enzymatic processes in which pyrimidine dimers are monomerized by UV and visible light. Two such non-enzymatic processes are (a) photolysis of dimers resulting from direct absorption of UV, and (b) sensitized monomerization involving charge transfer complexes. Several lines of evidence suggest that the mechanism of action of the PRE more closely resembles (b) than (a). Recent experiments on the PRE from E. coli reveal the presence of new long wavelength absorption which may indicate the presence of a ground state complex. The known ability of PRE to monomerize dimers of thymine, cytosine and uracil suggests that the carbonyl groups at 2 position of the pyrimidine ring may be important in the interaction between enzyme and dimer.     

PHOTOPHYSICS AND PHOTOCHEMISTRY OF 4-DIHYDROPYRIDINONES

Dihydropyridinones (DHP) react photochemically with olefins to form cycloaddition products. The reactions proceed through the lowest excited state T ₁ of the DHPs, with rate constants which depend on the olefin and can approach the diffusional limit in the most favourable cases. Intra-and inter-molecular sensitization and quenching have been investigated, as well as spectroscopic properties such as absorption and luminescence spectra, and in particular electron energy loss spectra which have established the energy of the reactive T₁, state as 295 kj mol^-1 (3.07 eV).     

PHOTOCHEMISTRY AND PHOTOPHYSICS OF GUANINE-CONTAINING DINUCLEOSIDES

Abstract— Yields and action spectra are reported for photochemistry. fluorescence. and total lumincscence at 405 nm due to UV excitation (240–300 nm) or dilutc (-0.1 mM ) solutions or guanosine 5–monophosphate (GMP) and the dinucleosides linking guanine with adenine (ApG and GpA). cytosine (CpG) and uracil (GpU) in neutral ethylene glycol-water (7:3) glasses at 140–165 K. Phosphorescence lifetimes were determined at 140 K. Less complete data are presented for GpC, UpG and dpGpT. Quantum yields for all three processes were usually found to increase as the excitation wavelength increases. Although intramolecular exciplex formation was not dominant under these conditions interactions were strong enough to frustrate attempts at interpretation of results within the Forster very weak coupling framework. There is evidence that the GMP photochemistry proceeds from the triplet state. Surprisingly, this photochemistry is not quenched in ApG, GpA, and dpGpT at 163 K although the adenine (A) and thymine (T) moieties are known to have lower triplet states. At 140 K the phosphorescence from ApG and GpA was entirely characteristic of A but both G and T components were observed from dpGpT.     

ACRYLAMIDE QUENCHING OF TRYPTOPHAN PHOTOCHEMISTRY AND PHOTOPHYSICS

Studies of acrylamide quenching of tryptophan (Trp) fluorescence, photochemistry, and photoionization have been conducted. Quenching of Trp fluorescence in aqueous solution by addition of acrylamide in the concentration range 0.0-0.5 M was measured and resulted in a Stern-Volmer quenching constant of KSV = 21 +/- 3 M-1. Photolysis experiments were performed in which Trp was photolyzed at 295 nm in the presence of varying concentrations of acrylamide. The loss of Trp was monitored using reverse-phase high performance liquid chromatography (RP-HPLC) and was observed to follow first order kinetics. Production of N-formylkynurenine (NFK) was observed by RP-HPLC in irradiated Trp samples both in the presence and absence of added acrylamide. In addition, no new photochemical product was detected. This was taken as evidence that acrylamide did not alter the photochemical pathway but just reduced the reaction rate as expected for a physical quenching mechanism. Plotting the reciprocal of photolysis rate constant versus acrylamide concentration produced a Stern-Volmer constant for quenching of Trp photochemistry of KSV = 6 +/- 2 M-1. The KSV values for both fluorescence quenching and photolysis quenching were thus large, implying efficient quenching of both processes by acrylamide. Assuming an excited singlet state lifetime of 2.8 ns, the calculated second-order quenching rate constants for fluorescence and photolysis were kq = 7.5 x 10(9) and 2.1 x 10(9) M-1 s-1 respectively. The possible involvement of photoionization in the photolysis mechanism was investigated by studies of acrylamide quenching of voltage transients produced by xenon flash lamp excitation of Trp at aqueous/teflon or aqueous/mica interfaces.(ABSTRACT TRUNCATED AT 250 WORDS)     

PHOTOSENSITIZATION OF PYRIMIDINE DIMER SPLITTING BY A COVALENTLY BOUND INDOLE

Abstract— Photosensitized pyrimidine dimer splitting characterizes the enzymatic process of DNA repair by the DNA photolyases. Possible pathways for the enzymatic reaction include photoinduced electron transfer to or from the dimer. To study the mechanistic photochemistry of splitting by a sensitizer representative of excited state electron donors, a compound in which an indole is covalently linked to a pyrimidine dimer has been synthesized. This compound allowed the quantitative measurement of the quantum efficiency of dimer splitting to be made without uncertainties resulting from lack of extensive preassociation of the unlinked dimer and sensitizer free in solution. Irradiation of the compound with light at wavelengths absorbed only by the indolyl group (approximately 280 nm) resulted in splitting of the attached dimer. The quantum yield of splitting of the linked system dissolved in N₂0-saturated aqueous solution was found to be 0.04 ± 0.01. The fluorescence typical of indoles was almost totally quenched by the attached dimer. A splitting mechanism in which an electron is efficiently transferred intramolecularly from photoexcited indole to ground state dimer has been formulated. The surprisingly low quantum yield of splitting has been attributed to inefficient splitting of the resulting dimer radical anion. Insights gained from this study have important mechanistic implications for the analogous reaction effected by the DNA photolyases.     

In situ PYRIMIDINE DIMER DETERMINATION BY LASER CYTOMETRY

By using antiserum against pyrimidine dimers and argon-laser imaging microspectrofluorometry, we established a new method to determine UV-induced pyrimidine dimers and their repair in individual human cells. The method was sensitive enough to determine dimers induced by UV dose as low as 2 J/m2. Normal human cells repaired 50 and 60% of total damage within 8 and 24 h after UV irradiation (20 J/m2), but Xeroderma pigmentosum cells (complementation group A) were unable to repair any within the same period. Therefore, the method proved to be a quick, easy, sensitive and accurate means to determine pyrimidine dimers in situ.     

CF3Br的红外光化学和光物理

We present the optothermal and optoacoustic spectra of CF_3Br in TEA CO_2 laser 00°1—02°0 R branch. Three absorption peaks has been observed at R(14) line (1074.6 cm~(-1)), R(18) line (1077.3cm~(-1)), and R(22) line (1079.9cm~(-1)) respectively. The peak at R(22) coincides with the position of P branch of CF_3Br linear IR spectrum, but two peaks at R(14) and R(18) can be interpreted as the hot bands.We also studied the multiphoton dissociation of CF_3Br and carbon isotope seperation.     

PYRIMIDINE DIMER INDUCTION AND REMOVAL IN THE EPIDERMIS OF HAIRLESS MICE: INEFFICIENT REPAIR IN THE GENOME OVERALL AND RAPID REPAIR IN THE H-ras SEQUENCE

Excision repair of pyrimidine dimers was examined at the genome overall in three strains of hairless ( hr/hr ) and congenic wild-type mice, as well as in the expressed H- ras gene in hairless mice. The assay used a pyrimidine dimer-specific endonuclease from Micrococcus luteus and alkaline agarose gel electrophoresis. From 0 to 25% of endonuclease-sensitive sites were removed at the genome level in either hairy or hairless mice but about 50% were removed in the H- ras gene in hairless mice by 24 h after exposure to 5.4 J/cm² UV (290-400 nm) irradiation. No differences were observed in the repair capacity between hairy and hairless mice, thus eliminating defective DNA repair as the explanation for the greater susceptibility to UV carcinogenesis in hairless mice.     

EXCISION REPAIR OF UVR-INDUCED PYRIMIDINE DIMERS IN CORNEAL DNA

Abstract— We measured excision repair of ultraviolet radiation (UVR)-induced pyrimidine dimers in DNA of the corneal epithelium of the marsupial, Monodelphis domestica , using damage-specific nucleases from Micrococcus luteus in conjunction with agarose gel electrophoresis. We observed that 100 J ^-2 of UVR from aFS–40 sunlamp(280–400 nm) induced an average of 2.2 ± 0.2 times 10^-2 endonuclease-sensitive sites per kilobase (ESS/kb) (pyrimidine dimers) and that ∼ 50% of the dimers were repaired within 12 h after exposure. We also determined that an exposure of 400 J m^-2 was needed to induce comparable numbers of pyrimidine dimers (2.5 times 10^-2) in the DNA of skin of M. domestica in vivo . In addition, we found that 50% of the dimers were also removed from the epidermal cells of M. domestica within 12 h after exposure. A dose of 100 J m^-2 was necessary to induce similar levels of pyrimidine dimers (2.0 ± 0.2 times 10^-2) in the DNA of the cultured marsupial cell line Pt K2 ( Potorous tridactylus ).     

THERMAL REQUIREMENTS OF PHOTOSENSITIZED PYRIMIDINE DIMER SPLITTING

Abstract— A cis, syn -pyrimidine dimer (derived from thymine and orotate) covalently linked to 5-methoxyindole has been studied as a mechanistic model of photosensitized pyrimidine dimer splitting. In this dimer-indole, photoinitiated electron transfer to the dimer causes splitting in a manner that parallels the mechanism by which the DNA photolyases are thought to act. Dissolved in EPA (diethyl ether-isopentane-ethyl alcohol, 5: 5: 1, by vol) at room temperature, the dimer-indole exhibited indole fluorescence quenching and underwent splitting upon irradiation at 300 nm. In an EPA glass at 77 K, however, no splitting was detectable. To distinguish the effects of temperature and immobilization, photolysis experiments were performed on PMM [poly(methyl methacrylate)] films containing dimer-indole. In PMM at room temperature, dimer-indole underwent splitting when irradiated at 300 nm, which indicated that immobilization per se was not responsible for the failure of dimer-indole to split at low temperature. Furthermore, no splitting was observed when dimer-indole was irradiated in PMM at 77 K. These results imply that a step following photoinitiated, intramolecular electron transfer from indole to dimer has an insurmountable activation barrier at 77 K. The mechanistic implications for the photolyases are considered.     

p-AMINOBENZOIC ACID-SUNLAMP SENSITIZATION OF PYRIMIDINE DIMER FORMATION AND TRANSFORMATION IN HUMAN CELLS

Abstract In the presence of sunlamp radiation, p -aminobenzoic acid sensitizes pyrimidine dimer formation in the DNA of human skin fibroblasts. It also sensitizes the sunlamp-induced transformation of such cells to anchorage-independent growth.     

TRANSIENT INTERMEDIATES IN INTRAMOLECULARLY PHOTOSENSITIZED PYRIMIDINE DIMER SPLITTING BY INDOLE DERIVATIVES

Abstract— Intramolecularly photosensitized pyrimidine dimer splitting can serve as a model for some aspects of the monomerization of dimers in the enzyme-substrate complex composed of a photolyase and UV-damaged DNA. We studied compounds in which a pyrimidine dimer was covalently linked either to indole or to 5-methoxyindole. Laser flash photolysis studies revealed that the normally observed photoejection of electrons from the indole or the 5-methoxyindole to solvent was diminished by an order of magnitude for indoles with dimer attached (dimer-indole and dimer-methoxyindole). The fluorescence lifetime of dimer-indole in aqueous methanol was 0.85 ns, whereas that of the corresponding indole without attached dimer (tryptophol) was 9.7 ns. Similar results were obtained for the dimer-methoxyindole (0.53 ns) and 5-methoxytryptophol (4.6 ns). The quantum yield of dimer splitting for the dimer-methoxyindole (φ₂₈₇K7 = 0.08) was only slightly greater than the value found earlier for the dimer bearing the unsubstituted indole (4>_2K7= 0.04). Transient absorption spectroscopy also revealed lower yields of indole radical cations following laser flash photolysis of dimer-indole compared to the indole without attached dimer. Dimer-methoxyindole behaved similarly. These results are interpreted in terms of an enhanced rate of radiationless relaxation of the indole and methoxyindole excited singlet states in dimer-indoles. The possible quenching of the indole and methoxyindole excited states via electron abstraction by the covalently linked dimer is discussed.     

PHOTO-CIDNP STUDY OF PYRIMIDINE DIMER SPLITTING I: REACTIONS INVOLVING PYRIMIDINE RADICAL CATION INTERMEDIATES

The light-induced splitting of pyrimidine dimers was studied using the electron acceptor anthraquinone-2-sulfonate (AQS) as a photosensitizer. To this end, photochemically induced dynamic nuclear polarization (photo-CIDNP) experiments were performed on a series of pyrimidine monomers and dimers. The CIDNP spectra demonstrate the existence of both the dimer radical cation, which is formed by electron transfer from the dimer to the photoexcited sensitizer AQS*, and its dissociation product, the monomer radical cation. In spectra of 1,1′-trimethylene bridged cis,syn pyrimidine dimers, polarization is observed that originates from a spin-sorting process in the dimer radical pair. This points to a relatively long lifetime of the dimer radical cation involved, which is presumably due to stabilization by the trimethylene bridge. Polarization originating from a dimer radical pair is detected in the spectrum of trans,anti (1,3-dimethyluracil) dimer as well. The spectra of the bridged pyrimidines also demonstrate the reversibility of the dissociation of dimer radical cation into monomer radical cation, which is concluded from the observation of polarization in the dimer as a result of spin sorting in the monomer radical pair.     

THE PHOTOPHYSICS AND PHOTOCHEMISTRY OF THE NEAR-UV ABSORBING AMINO ACIDS-III. CYSTINE AND ITS SIMPLE DERIVATIVES

Abstract— The photochemistry of cystine and some of its analogues is reviewed. Topics discussed include the absorption spectra of disulfides, the photochemistry of alkyl disulfides and other more highly substituted disulfides including cystine, photooxidation of disulfides, and photochemical interactions of cystine derivatives with other molecules of biological interest.     

PHOTO-CIDNP STUDY OF PYRIMIDINE DIMER SPLITTING II: REACTIONS INVOLVING PYRIMIDINE RADICAL ANION INTERMEDIATES

A series of photo-CIDNP (chemically induced dynamic nuclear polarization) experiments were performed on pyrimidine monomers and dimers, using the electron-donor Nα-acetyltryptophan (AcTrp) as a photosensitizer. The CIDNP spectra give evidence for the existence of both the dimer radical anion, which is formed by electron transfer from the excited AcTrp* to the dimer, and its dissociation product, the monomer radical anion. The AcTrp spectra are completely different from those obtained with an oxidizing sensitizer like anthraquinone-2-sulfonate, because of different unpaired electron spin density distributions in pyrimidine radical anion and cation. In the spectra of the anti (1,3-dimethyluracil) dimers, polarization is detected that originates from a spin-sorting process in the dimer radical pair, pointing to a relatively long lifetime of the dimer radical anions involved. Although the dimer radical anions of the 1,1′-trimethylene-bridged pyrimidines may have a relatively long lifetime as well, their protons have only very weak hyperfine interaction, which explains why no polarization originating from the dimer radical pair is detected. In the spectra of the bridged pyrimidines, polarized dimer protons are observed as a result of spin sorting in the monomer radical pair, from which it follows that the dissociation of dimer radical anion into monomer radical anion is reversible. A study of CIDNP intensities as a function of pH shows that a pH between 3 and 4 is optimal for observing monomer polarization that originates from spin-sorting in the monomer radical pair. At higher pH the geminate recombination polarization is partly cancelled by escape polarization arising in the same product.     

MATHEMATICAL ASPECTS OF THE ULTRAVIOLET PHOTOCHEMISTRY OF PYRIMIDINE DINUCLEOSIDE PHOSPHATES

Abstract A mathematical formulation of the Kinetics of dimner and hydrate formation in pyrimidine dinucleosides upon ultraviolet (u.v.) radiatin is presented. The formulation allows for the possibilities of forming a number of types of dimers each with different cross sections for formtion and reversal, and of forming photo-irreversible products such as hydrates. The general differential equations descrbinjg the kinetics are solved in terms of a series of exponential terms which are functions of all the cross sections. The way in which any given species of hotoproduct is dependent upon the cross sections is illustrted in several examples. The results have been used to predict the maximum amount of dirner which may be produced for a given set of cross sections and the exposure at which this maximum is expected. The conditions under which dimers may be detected in a mixture of parent and dimer upon re-irradiation at short wavelengths are examined in terms of the differential equations.