The (6-4) photoproduct is an important determinant of the lethal and mutagenic effects of UV irradiation of biological systems. The removal of this lesion appears to correlate closely with the early DNA repair responses of mammalian cells, including DNA incision events, repair synthesis and removal of replication blocks. The processing of (6-4) photoproducts and cyclobutane dimers appears to be enzymatically coupled in bacteria and most mammalian cell lines examined (i.e. a mutation affecting the repair of one lesion also often affects the other), although exceptions exist in which repair capacity may be evident for one photoproduct and not the other (e.g. UV61 and the XP revertant cell line). These differences in the processing of the two photoproducts in some cell lines of human and rodent origin suggest that in mammalian cells, different pathways for the repair of (6-4) photoproducts and cyclobutane dimers may be used. This observation is further supported by pleiotropic repair phenotypes such as those observed in CHO complementation class 2 mutants (e.g., UV5, UVL-1, UVL-13, and V-H1). Indirect data, from HCR of UV irradiated reported genes and the cytotoxic responses of UV61, suggest that the (6-4) photoproduct is cytotoxic in mammalian cells and may account for 20 to 30% of the cell killing after UV irradiation of rodent cells. Cytotoxicity of the (6-4) photoproduct may be important in the etiology of sunlight-induced carcinogenesis, affecting mutagenesis as well as tumorigenesis. The intricate photochemistry of the (6-4) photoproduct, its formation and photoisomerization, is in itself extremely interesting and may also be relevant to sunlight carcinogenesis. The data reviewed in this article support the notion that the (6-4) photoproduct and its Dewar photoisomer are important cytotoxic determinants of UV light. The idea that the (6-4) photoproduct is an important component in the spectrum of UV-induced cytotoxic damage may help clarify our understanding of why rodent cells survive the effects of UV irradiation as well as human cells, without apparent cyclobutane dimer repair in the bulk of their DNA. The preferential repair of cyclobutane dimers in essential genes has been proposed to account for this observation (Bohr et al., 1985, 1986; Mellon et al., 1986). The data reviewed here suggest that understanding the repair of a prominent type of noncyclobutane dimer damage, the (6-4) photoproduct, may also be important in resolving this paradox.

The biology of the (6-4) photoproduct.

The frequency of UV-induced mutations at specific nucleotides along the lacI gene of Escherichia coli varies by as much as 80-fold1. The spectrum of mutations to amber, ochre, and UGA chain-terminating codons includes five hotspots accounting for over 30% of these UV-induced nonsense mutations1. The simplest explanation for the hotspot phenomenon is that the mutation frequency reflects the frequency of UV-induced DNA damage at the mutation site. To test this possibility, we measured the distribution, in a 380-base pair (bp) region of the lacI gene containing the nonsense mutation hotspots, of UV-induced cyclobutane pyrimidine dimers and pyrimidine–pyrimidine (6-4) photoproducts. The latter (also called the PydC lesion) is a UV-induced DNA lesion which occurs predominantly at TC and CC sequences2. The results reported here reveal the presence of UV-induced base damage hotspots which include the nonsense mutation hotspots. There is a linear relationship between base damage incidence and mutation incidence. The results suggest that the UV-induced pyrimidine–pyrimidine (6-4) lesion may be mutagenic.

UV-induced mutation hotspots occur at DNA damage hotspots

Publisher Summary This chapter is concerned with the structures and interactions of purines and pyrimidines in the crystalline state. However, because of their close similarity to the pyrimidines, it also discusses the crystal structure of the barbiturates. The chapter reviews the structure arid conformation of the sugar and the phosphate components of nucleosides, nucleotides, and nucleic acids, and their steric relationship to the purines and pyrimidines. It discusses relevance of single-crystal analysis of purine–pyrimidine interactions to the fiber analysis of polynucleotides. The chapter provides an overview on X-ray diffraction method. It presents a complete tabulation of the molecular geometry of the reliable structures for which there is three-dimensional diffraction information. It draws the conclusion that the structures of the individual purine and pyrimidine bases retain a fair amount of constancy in a variety of different crystal lattices. The chapter also focuses on the H-bonds that are formed between the purines and pyrimidines. In contrast to the narrow range of covalent bond lengths that are found within the molecules, there is considerable variation in both the length of H-bonds and their angles, a point that is emphasized in this presentation. As there is an appreciable range in the geometry of H-bonded interactions, it is likely that a greater variety of stable structures can be formed. An important variable in this connection is the variation in H-bond lengths, as the length of this bond characterizes the energy of H-bonding interactions in part. It reviews that purine and pyrimidine crystalIography underlines the diversity of the H-bonding potentiality of the nucleic acid bases; it is likely that this diversity has been utilized fully by nature in the evolution of the information-transferring system.

The crystal structures of purines, pyrimidines and their intermolecular complexes.

Cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts ((6-4)photoproducts) are the two major classes of cytotoxic, mutagenic and carcinogenic DNA photoproducts produced by ultraviolet light irradiation of cells. The phenomenon of photoreactivation, the reduction of the lethal and mutagenic effects of ultraviolet radiation by simultaneous or subsequent irradiation with near ultraviolet or visible light, has been identified in several organisms and in some cases the enzymes that catalyse this process have been characterized in sufficient detail. CPDs are the only known substrate for the photoreactivating enzymes so far analysed and enzymatic photoreactivation of (6-4)photoproducts has not yet been reported. We report here that an enzyme that catalyses the light-dependent repair of (6-4)photoproduct exists in Drosophila melanogaster. This is, to our knowledge, the first report of such photoreactivating activity specific for (6-4)photoproducts in any organism.

A new photoreactivating enzyme that specifically repairs ultraviolet light-induced (6-4)photoproducts

The ability of cells to maintain genomic integrity is vital for cell survival and proliferation. Lack of fidelity in DNA replication and maintenance can result in deleterious mutations leading to cell death or, in multicellular organisms, cancer. The purpose of this review is to discuss the known signal transduction pathways that regulate cell cycle progression and the mechanisms cells employ to insure DNA stability in the face of genotoxic stress. In particular, we focus on mammalian cell cycle checkpoint functions, their role in maintaining DNA stability during the cell cycle following exposure to genotoxic agents, and the gene products that act in checkpoint function signal transduction cascades. Key transitions in the cell cycle are regulated by the activities of various protein kinase complexes composed of cyclin and cyclin-dependent kinase (Cdk) molecules. Surveillance control mechanisms that check to ensure proper completion of early events and cellular integrity before initiation of subsequent events in cell cycle progression are referred to as cell cycle checkpoints and can generate a transient delay that provides the cell more time to repair damage before progressing to the next phase of the cycle. A variety of cellular responses are elicited that function in checkpoint signaling to inhibit cyclin/Cdk activities. These responses include the p53-dependent and p53-independent induction of Cdk inhibitors and the p53-independent inhibitory phosphorylation of Cdk molecules themselves. Eliciting proper G1, S, and G2 checkpoint responses to double-strand DNA breaks requires the function of the Ataxia telangiectasia mutated gene product. Several human heritable cancer-prone syndromes known to alter DNA stability have been found to have defects in checkpoint surveillance pathways. Exposures to several common sources of genotoxic stress, including oxidative stress, ionizing radiation, UV radiation, and the genotoxic compound benzo[a]pyrene, elicit cell cycle checkpoint responses that show both similarities and differences in their molecular signaling.

Cell cycle control, checkpoint mechanisms, and genotoxic stress.

A product isolated from thymine irradiated with ultraviolet light in frozen aqueous solution undergoes dehydration on heating with acids. As judged by elemental analysis, mass, ultraviolet, infrared, and nuclear magnetic resonance spectra, the most probable structures for this compound and its dehydration product, respectively, are 5-hydroxy-6-4'-[5''-methylpyrimidin-2'-one]-dihydrothymine and 6-4'-[5'-methylpyrimidin-2'-one]-thymine. Apparently, this compound is a thymine-thymine adduct and presumably is formed through the rearrangement of an initial photoproduct. Both compounds are closely related to 6-4'-[pyrimidin-2'-one]-thymine which has been isolated from acid hydrolyzates of ultraviolet-irradiated DNA and supposedly is derived from cytosine-thymine adduct. Formation of such adducts between pyrimidine bases is apparently a common photoreaction and may be important to the study of the photochemistry and photobiology of nucleic acids.

https://science.sciencemag.org/content/sci/160/3824/186.full.pdf

Thymine-Thymine Adduct as a Photoproduct of Thymine

A new thymine-derived product was separated from DNA irradiated with utlraviolet light in vitro and in vivo. This compound was mistaken to be thymine homodiner (T=T) by other workers because it is chromatographically indistinguishable from T=T in most eluents. It has absorbancy maximums at 312, 312, and 300 millimicrons in neutral, pH 2, and pH 11 aqueous solutions, respectively. When it is irradiated in aqueous solution with wavelengths of 360 and 313 millimicrons its spectrum reverts to one similar to that of thymine. Therefore, at least three thymine-derived products can be detected in ultraviolet irradiated DNA, namely the homodimer, a material with absorbancy maximum at 312 millimicrons, and a "minor" product suggested by others to be a dimer of cytosine and thymine. In cells, the latter two are formed in aboult equal amounts. While these three products were shown to exist in the acid hydrolyzates of ultraviolet irradiated DNA, a material with absorbancy maximum at about 310 millimicrons was demonstrated to form in ultraviolet irradiated DNA without further treatment. The magnitude of this spectral increase varied directly with the incrcase in the adenine-thymine contents in the DNA as shlown by differential transmittance spectra of the irradiated Micrococcus lysodeikticus, calf thymus, Bacillus cereus, and Hemophilus influenzae DNA.

https://science.sciencemag.org/content/sci/156/3777/955.full.pdf

Ultraviolet irradiation of dna in vitro and in vivo produces a third thymine-derived product.

Thymine-thymine adduct is a product isolated from thymine irradiated with ultraviolet light in frozen aqueous solution. This compound is presumably formed through the rearrangement of an initial photoproduct. Single crystal x-ray diffraction analysis has confirmed the molecular formula of the adduct, 5-hydroxy-6-49-(59-methylpyrimid-29-one)-dihydrothymine, except for the possibility of a hydrogen atom on the 39 nitrogren rather than the 19 nitrogen, and has established the stereoconfiguration of the molecule.

https://science.sciencemag.org/content/sci/164/3876/183.full.pdf

A. J. Varghese

Papers

Thymine-Thymine Adduct as a Photoproduct of Thymine

Ultraviolet irradiation of dna in vitro and in vivo produces a third thymine-derived product.

Crystal and Molecular Structure of a Thymine-Thymine Adduct