Excited-State Dynamics in the RNA Nucleotide Uridine 5'-Monophosphate Investigated Using Femtosecond Broadband Transient Absorption Spectroscopy.

Damage to RNA from ultraviolet radiation induces chemical modifications to the nucleobases. Unraveling the excited states involved in these reactions is essential; however, investigations aimed at understanding the electronic-energy relaxation pathways of the RNA nucleotide uridine 5'-monophosphate (UMP) have not received enough attention. In this Letter, the excited-state dynamics of UMP is investigated in aqueous solution. Excitation at 267 nm results in a trifurcation event that leads to the simultaneous population of the vibrationally excited ground state, a long-lived 1nπ* state, and a receiver triplet state within 200 fs. The receiver state internally converts to the long-lived 3ππ* state in an ultrafast time scale. The results elucidate the electronic relaxation pathways and clarify earlier transient absorption experiments performed for uracil derivatives in solution. This mechanistic information is important because long-lived nπ* and ππ* excited states of both singlet and triplet multiplicities are thought to lead to the formation of harmful photoproducts.

Detecting the ability of viral, bacterial and eukaryotic replication proteins to track along DNA.

The phage T4 gene 45 protein (gp45), Escherichia coli beta and the eukaryotic proliferating cell nuclear antigen (PCNA) function in replication as processivity factors of their corresponding DNA polymerases. The T4 gp45 also functions as the transcriptional activator that connects expression of viral late genes to DNA replication. DNA tracking is an essential component of the replication and transcription regulatory functions of T4 gp45. The ability of gp45, beta and PCNA to track along DNA has been analyzed by photocrosslinking. Each of these proteins must be loaded onto DNA by a species-specific assembly factor. For gp45 and beta, the density of traffic along DNA is determined by a dynamic balance between continuous protein loading and unloading, and is also dependent on interaction with the conjugate single-stranded DNA binding protein.

Characterization of free radicals in gamma-irradiated polycrystalline uridine 5'-monophosphate: a study combining ESR, spin-trapping and HPLC.

Free radicals generated in gamma-irradiated polycrystalline uridine 5'-monophosphate (5'-UMP) were studied by ESR, spin-trapping and high-performance liquid chromatography (HPLC). After gamma-irradiation at 0 degree C (70kGy), poly-crystalline 5'-UMP was dissolved in an anaerobic aqueous solution of 2-methyl-2-nitrosopropane as a spin trap at room temperature. Since an ESR spectrum consisting of several components was observed immediately after irradiation, these components were separated with reverse-phase HPLC in the ion-suppression mode and again analysed by ESR spectrometry. Although HPLC ultimately gave four spin-adducts, one component that was originally present disappeared during HPLC. Spin adducts due to two types of C6 radicals were identified. One of these was thought to be formed by electron addition and subsequent protonation at the C6 position, and the other was presumed to be produced by electron addition and subsequent protonation at the O4 position. The spin adducts derived from the C5 and C5' radicals were also identified. The spin adduct that disappeared during HPLC was thought to correspond to the C4'-centred radical. Computer simulation of ESR spectra was carried out to estimate the hyperfine splitting constants.

Ultraviolet irradiation of nucleic acids: formation, purification, and solution conformational analyses of the cis-syn and trans-syn photodimers of UpU.

Uridylyl(3'-5')uridine (UpU) is subjected to aqueous acetone photosensitized radiation with sunlamps. These irradiation conditions form only cyclobutane-type photodimers. Purification of a specific configurational photodimer is accomplished by using C-18 reverse-phase high-performance liquid chromatography. Multinuclear NMR analysis is used to analyze photoproduct formation and to determine conformational features of these photodimers. Four photodimers are identified, with the cis-syn isomer predominant. The cis-syn and trans-syn photodimers of UpU exhibit markedly different furanose and exocyclic bond conformations. A comparison of the properties of the cis-syn dimers of UpU with those of dTpdT reveal many similar conformational features but also some that are different.

Gamma-radiolysis of poly(U) in aqueous solution. The role of primary sugar and base radicals in the release of undamaged uracil.

p6e release of undamaged uracil has been measured after gamma-irradiation of aqueous solutions of poly(U). By varying the experimental conditions, the respective efficiencies with which OH, H and e-aq release uracil were found to be 51, 28 and about 3 per cent. From the determination of G(H2) in acid solutions it is concluded that greater than or equal to 95 per cent of the H atoms add to the base moiety. It is proposed that the H-adduct radicals (and similarly the OH-adduct radicals) react with the sugar moiety of a different nucleotide. Subsequent reactions of the sugar radicals so formed lead to the release of undamaged uracil. In N2O-saturated solutions the addition of tetranitromethane (TNM) causes a sharp drop in released uracil from G = 2.9 to G = 0.19. The strong suppression of uracil release by TNM is presumably due to its scavenging of the major base radical with the remaining G(uracil release) being due to a direct attack of OH at the sugar moiety. In contrast, G(uracil release) from 5'-UMP gamma-irradiated under N2O is only 0.39, dropping to 0.31 in the presence of TNM. This indicates the important role of the polymeric structure in the mechanism leading to efficient base release. These results on base release are compared with reported data on strand breakage.

Radiolysis of nucleosides in aqueous solutions: base liberation by the base attack mechanism.

On the radiolysis of uridine and some other nucleosides in aqueous solution, a pH-dependent liberation of uracil or the corresponding base was found. e-aq and HO-.2 gave no freed bases, although many oxidizing radicals, including OH, Cl-.2, Br-.2, (CNS)-.2 and SO-.4, did cause the release of unaltered bases, depending on the pH of the solutions. The base yields were generally high at pH greater than or equal to 11, with the exception of SO-.4, which gave a rather high yield of uracil (from uridine) even in the pH region of less than or equal to 11. The pattern of the base formation against pH varied with the reacting radical, but was the same for the different nucleosides. With regard to the OH radical system, bases are considered to be formed after H-abstraction of the sugar moiety at low pH. The other oxidizing radicals, however, may cause base release by attack at the base. It is possible that O-, present at high pH as the dissociated form of OH, may act partly as an oxidizing radical. A plausible mechanism of 31'-radical formation is discussed.

Uridine 5'-phosphate photohydrate formation in irradiated Escherichia coli 30S ribosomes: photochemistry and relation to ribonucleic acid-protein cross-linking.

Irradiation of aqueous buffered solutions of Escherichia coli 30S ribosomes with doses of 254-nm radiation greater than 10(19) quanta causes formation of uridine 5'-phosphate (UMP) photohydrates in ribosomal 16S RNA (rRNA). The number of molecules of UMP photohydrate formed at doses less than 2 x 10(20) quanta is linearly dependent on dose of absorbed 254-nm radiation. Maximum UMP photohydrate formation is dependent on initial ribosome concentration. When solutions containing 1 A260 unit of 30S ribosomes/mL were irradiated with greater than 2 x 10(20) quanta of 254-nm radiation, maximum photohydrate formation was equal to 47 residues/ribosome. Irradiation of solutions containing 2 A260 units/mL with greater than 7 x 10(20) quanta caused formation of 102 UMP photohydrates/ribosome. These values correspond to conversion of either 15 or 33%, respectively, of the total UMP content of 30S ribosome 16S rRNA to photohydrates. Target theory analysis of UMP photohydration in 30S ribosomes showed that UMP photohydrates are formed by single-hit kinetics from two photochemically distinct precursors. Of the total 16S rRNA UMP residues, 10% was included in the most rapidly (low dose) reacting fraction. The respective photohydration cross sections are 0.014 (low dose) and 0.0095 cm2/muEinstein (high dose) for ribosome solutions containing 2 A260 units/mL. UMP photohydrate content of irradiated 30S ribosomes was compared with that of previous data for the extent of RNA-protein cross-linking at equivalent doses of absorbed 254-nm radiation. This comparison showed that at least two UMP photohydrates form per RNA-protein cross-linking event in 30S ribosomes irradiated with a dose of 254-nm radiation (1.5 x 10(19) quanta), which causes cross-linking of only three ribosomal proteins to 16S rRNA.

Free radicals from X-irradiated single crystals of uridine-5'-phosphate disodium salt.

X-irradiation of single crystals of uridine-5'-phosphate (disodium salt) between 10 and 300 K as well as storage of irradiated crystals at 300 K produces at least seven different radical species. Between 10 and 77 K, the uracil base anion and a secondary alkoxy radical at the ribose-O3'-site are formed. The latter transforms into a C5'-centred alkylphosphate species between 110 and 130 K which in turn decays between 180 and 220 K under formation of a base 5-yl hydrogen addition radical. Irradiation at 300 K additionally produces the base-located 6-yl radical together with a radical tentatively assigned to the doubly protonated base anion. Storage of crystals for several months results in decay of most of these species leaving a radical possibly located at c5' of the ribose. The spectral parameters of these radicals are given and discussed.

An e.s.r. study of stable radicals in gamma-irradiated single crystals of uridine 5'-phosphate (Na salt).

Electron spin resonance has been used for the study of the radical in gamma-irradiated single crystals of 5'-UMP (sodium salt). Six types of radicals have been identified at room temperature. They are formed by addition of H atoms to C5, C6, 02 and 04, and by an OH addition to C5 of the uracil ring. Five of these radicals have an appreciable spin density on C6. The radicals with the addition to C5 or C6 are less stable than than the essentially planar radicals with the H addition to 02 and 04.