R. B. Setlow

Effects of ultraviolet light on thymidine dinucleotide and polynucleotide

Abstract 1. 1. The absorbance changes in dithymidylic acid and in polythymidylic acid produced by ultraviolet irradiation have been interpreted in terms of the reversible formation of dimers between adjacent thymine residues. The formation of dimers, breakage of dimers, and steady-state fraction of dimers are all wavelength dependent. 2. 2. The action spectra for dimer formation are approximately the same as the absorption spectrum of thymine. The quantum yields are approx. 0.01 for dithymidylic-acid and approx. 0.02 for polythymidylic acid. 3. 3. The action spectra for dimer breakage are the same for both polymers and are similar to that for breaking the thymine dimer. The quantum yield is approx. 1. 4. 4. The steady-state fraction of dimers is approx. 70% for 280 mμ and approx. 15% for 240-mμ irradiation.

DNA Chain Elongation and Joining in Normal Human and Xeroderma Pigmentosum Cells after Ultraviolet Irradiation

DNA synthesized in human cells after ultraviolet (UV) irradiation is made in segments of lower molecular weight than in unirradiated cells. Within several hours after irradiation these smaller units are both elongated and joined together. This repair process has been observed in normal human fibroblasts, HeLa cells, and fibroblasts derived from three types of xeroderma pigmentosum patients-uncomplicated with respect to neurological problems, complicated (de Sanctis-Cacchione syndrome), and one with the clinical symptoms of xeroderma pigmentosum but with normal repair replication. The ability of human cells to elongate and to join DNA strands despite the presence of pyrimidine dimers enables them to divide without excising the dimers present in their DNA. It may be this mechanism which enables xeroderma pigmentosum cells to tolerate small doses of UV radiation.

Recovery of the ability to synthesize DNA in segments of normal size at long times after ultraviolet irradiation of human cells.

DNA synthesized in human cells within the first hour after ultraviolet (UV) irradiation is made in segments of lower molecular weight than in nonirradiated cells. The size of these segments approximates the average distance between pyrimidine dimers in the parental DNA. This suggests that the dimers interrupt normal DNA synthesis and result in gaps in the newly synthesized DNA. However, DNA synthesized in human cells at long times after irradiation is made in segments equal or nearly equal to those synthesized by nonirradiated cells. The recovery of the ability to synthesize DNA in segments of normal size occurs in normal human cells, where the dimers are excised, and also in cells of the human mutants xeroderma pigmentosum (XP), where the dimers remain in the DNA. This observation implies that the pyrimidine dimer may not be the lesion that causes DNA to be synthesized in smaller than normal segments.

PHOTOPRODUCTS IN DNA IRRADIATED IN VIVO

Abstract— Ultraviolet irradiation of DNA in vitro and in vivo results in the formation of many different types of photoproducts. The general procedure for correlating such photoproducts with photo‐biological effects is outlined and is applied for cyclobutane pyrimidine dimers and for spore photoproducts.

Repair of Chemical Damage to Human DNA

Our experiments and the substance of this chapter deal with chemical mutagens and chemical carcinogens and how they affect human cells in vitro. In particular, we assay damage to human DNA and the extent to which it is repaired after treatment with such chemicals. Thus, for our own purposes, we tend to consider all these chemicals as DNA-damaging agents, irrespective of their actions in other systems as mutagens, carcinogens, or both. To point up the importance of repair studies, we will first discuss the sequence of molecular events in hypothetical experiments that do assay mutation and/or carcinogenesis, and we will indicate at what point the nature of the primary lesion and the extent of its repair become matters of prime interest.

DNA repair in potorous tridactylus

The DNA synthesized shortly after ultraviolet (UV) irradiation of Potorous tridactylis (PtK) cells sediments more slowly in alkali than that made by nonirradiated cells. The size of the single-strand segments is approximately equal to the average distance between 1 or 2 cyclobutyl pyrimidine dimers in the parental DNA. These data support the notion that dimers are the photoproducts which interrupt normal DNA replication. Upon incubation of irradiated cells the small segments are enlarged to form high molecular weight DNA as in nonirradiated cells. DNA synthesized at long times ( approximately 24 h) after irradiation is made in segments approximately equal to those synthesized by nonirradiated cells, although only 10-15% of the dimers have been removed by excision repair. These data imply that dimers are not the lesions which initially interrupt normal DNA replication in irradiated cells. In an attempt to resolve these conflicting interpretations, PtK cells were exposed to photoreactivating light after irradiation and before pulse-labeling, since photoreactivation repair is specific for only one type of UV lesion. After 1 h of exposure approximately 35% of the pyrimidine dimers have been monomerized, and the reduction in the percentage of dimers correlates with an increased size for the DNA synthesized by irradiated cells. Therefore, we conclude that the dimers are the lesions which initially interrupt DNA replication in irradiated PtK cells. The monomerization of pyrimidine dimers correlates with a disappearance of repair endonuclease-sensitive sites, as measured in vivo immediately after 1 h of photoreactivation, indicating that some of the sites sensitive to the repair endonuclease (from Micrococcus luteus) are pyrimidine dimers. However, at 24 h after irradiation and 1 h of photoreactivation there are no endonuclease-sensitive sites, even though approximately 50% of the pyrimidine dimers remain in the DNA. These data indicate that not all pyrimidine dimers are accessible to the repair endonuclease. The observation that at long times after irradiation DNA is made in segments equal to those synthesized by nonirradiated cells although only a small percentage of the dimers have been removed suggests that an additional repair system alters dimers so that they no longer interrupt DNA replication.

Excision of pyrimidine dimers from irradiated deoxyribonucleic acid in vitro

Abstract An extract of cells of Micrococcus lysodeikticus is able to excise thymine-thymine and cytosine-thymine dimers from ultraviolet-irradiated deoxyribonucleic acid. The rate of excision is greater than the rate of hydrolysis of the deoxyribonucleic acid. Dimers excised from the deoxyribonucleic acid appear as parts of acid-soluble oligonucleotides which seem to have one terminal phosphate group. The excision in vitro is similar to the reaction observed in vivo in ultraviolet-resistant cells and is probably one of the steps in the dark repair of the biological activity of irradiated deoxyribonucleic acid in vitro .

Contribution of Dimers containing Cytosine to Ultra-violet Inactivation of Transforming DNA

BEUKERS1 has shown that dimers containing thymine in DNA irradiated at 2537 Å can be eliminated by further irradiation at 2537 Å in the presence of proflavine, and later work2 has confirmed his hypothesis that the elimination represents splitting of the dimers. We have shown that the biological inactivation of ultra-violet-irradiated transforming DNA from Haemophilus influenzae may similarly be reversed by irradiation in solutions containing proflavine. An investigation of the wavelength dependence of this reactivation in the presence of proflavine, together with information on the reversal of different types of pyrimidine dimers2, has made it possible to evaluate the role of dimers containing cytosine in inactivation.

Ergosterol and Substitutes for the Ultraviolet Radiation Requirement for Conidia Formation In Stemphylium Solani

Stemphylium solani preconidiophores respond by subsequent pigmentation and conidia production to treatment with certain solvents and carriers alone or with added ergosterol. Treatment consisted of flooding 7-day-old cultures grown in the dark at 21 C on V-8 juice 1.5% agar with 4 ml of solution so that preconidiophores are wetted or soaked for 24 hr prior to deflooding and dark incubation for another 24 hr. Conidia were induced to form by 1-10% DMSO in 0.1 M KH2PO4 and by 2% ethanol. Ergosterol added to carriers or solvents increased conidial formation 2to 5-fold over that in controls. The morphology and pigmentation of conidiophores and conidia were similar to those produced by ultraviolet radiation at appropriate doses. A possible explanation is that free ergosterol is involved in the conidial formation in S. solani.

THE EFFECT OF PROFLAVINE PLUS VISIBLE LIGHT ON THE DNA OF HUMAN CELLS

We undertook the experiments reported here because we were interested in the effect of the therapy utilizing proflavine plus visible light on normal human cells in the vicinity of the target herpes-infected ones. The experiments reported here deal solely with uninfected human cells. We used an in uitro system to approximate-at least qualitatively-the treatment in uiuo and the subsequent effects on cellular DNA. It has been shown that dark repair of dye-light damage to E. coli and phage DNA occurs (Harm, 1968; Imray and MacPhee, 1973).