Kendric C. Smith

Dose dependent decrease in extractability of DNA from bacteria following irradiation with ultraviolet light or with visible light plus dye.

Abstract In 1928 Gates pointed out the probable relation between the bactericidal effectiveness of the various wave lengths of ultraviolet light (U.V.) and the absorption of U.V. by deoxyribonucleic acid (DNA). In 1941, Hollaender et al. described changes in the macromolecular properties of DNA due to U.V. irradiation. Sinsheimer and Hastings (1949) reported that uracil and cytidylic acid can undergo reversible hydration in U.V. Setlow and Doyle (1954) and Baranowska and Shugar (1960) have reported the U.V. crosslinking of dry films of DNA. In 1960 Beukers and Berends reported that U.V. can cause the dimerization of thymine and Wacker et al. (1960) found this to occur in vivo (presumably intrachain). Marmur and Grossman (1961) have reported the interchain crosslinking of DNA by U.V. The present report deals with a new phenomenon: a dose dependent decrease in the extractability of DNA when bacteria are irradiated with U.V. This response is analytically many times more sensitive than in vivo thymine dimer formation ( Smith, 1962 ). This phenomenon also occurs when the bacteria are sensitized with acridine orange and exposed to visible light, but does not seem to be caused by x-irradiation.

Repair of radiation-induced damage in Escherichia coli: I. Effect of rec mutations on post-replication repair of damage due to ultraviolet radiation☆☆☆

Abstract The rec mutants of Escherichia coli K12 are more sensitive to ultraviolet radiation than corresponding rec + derivatives. We have tested several of these rec mutants for post-replication repair after irradiation with ultraviolet light. The mutants carried recA13 , recA56 , recB21 and recC22 . Cultures growing exponentially were exposed to ultraviolet light (63 ergs/mm 2 ) and then labeled for ten minutes with [ 3 H]thymidine. The radioactive medium was removed and the culture divided into two portions, one of which was chilled while the other was reincubated in non-radioactive medium for various times. The DNA from the cells was then analysed on alkaline sucrose gradients. Labeled DNA from irradiated cells which had not been reincubated sedimented as short molecules. After reincubation, DNA from the recA mutants still behaved as short molecules, while DNA from rec + , recB and recC cells assumed a position similar to that of DNA from unirradiated control cells. These results indicate that the two recA mutants tested were defective in their capacity for post-replication of damage induced by ultraviolet radiation. However, no deficiency in the recB and recC mutants was detected under the conditions used. Even in uvr + cells, which are capable of excision-dependent repair, the formation of short molecules could be demonstrated immediately after irradiation. However, after a 40-minute post-irradiation incubation before labeling with [ 3 H]thymidine, only “normal-sized” DNA was formed. In contrast, in a uvr − strain, short molecules were still formed after a 90 minute post-irradiation incubation. These results indicate that excision-dependent repair can remove the lesions that lead to the synthesis of short pieces of DNA after ultraviolet-irradiation. Similarly, the exposure of ultraviolet cells to conditions of photoreactivation (which repairs pyrimidine dimers in situ ) before labeling greatly diminished but did not completely eliminate the synthesis of short pieces of DNA.

Effect of Halogenated Pyrimidines on Radiosensitivity of E. coli

The incorporation of halogcnated thymine and thymidine analogs into E.coli DNA during log-phase growth, though relatively nontoxic per se, was associated with a significant increase in sensitivity of the bacterial cells to x and uv irradiation. Although incorporation was linear with time from 2 to 10 hours of incubation, significant radiosensitization was not observed untiI 4 hours (l7% replacement of thymine by 5-bromouracil) and was maximal at the end of log-phase growth (40 to 50% replacement), despite a further increment of incorporation during stationary phase, Labeling of only one strand of the DNA double helix was sufficient to confer at least half-maximal radiosensitization. The implications of the data for clinical radiotherapy are discussed.

PHOTOCHEMICAL REACTIONS OF THYMINE, URACIL, URIDINE, CYTOSINE AND BROMOURACIL IN FROZEN SOLUTION AND IN DRIED FILMS*

Abstract— The photochemistry of uracil, uridine, cytosine, thymine and broinouracil has been investigated in frozen aqueous solution and in dried films. Essentially the same photoproducts were obtained in the two conditions; however, the yield of photoproducts was considerably greater in frozen solution. Uracil forms a dimer which can exist in two forms. Some kinetic data are presented for the interconversion of these two forms, The mixed dimer of thymine and uracil can also exist in two forms. Uridine forms only one acid stable photoproduct and does not appear to form mixed photoproducts under the conditions used. Two new photoproducts of thymine other than the dimer are described. Cytosine was at first considered to be completely inert hut using more sensitive detecting equipment it has recently been found to form uracil dinier as a result of dinierization and deamination. The most remarkable response was shown by bromouracil. When irradiated by itself it formed no photoproducts but when irradiated in the presence of uracil, uridine, cytosine or NaOH it formed many photoproducts. Most of these products were devoid of bromide, but two still contained bromine. One of these has been identified as the mixed dimer of uracil and bromouracil while the other has been tentatively identified as the dimer of bromouracil. Dimers of thymine or bromouracil were not formed by X‐rays.

DNA POLYMERASE REQUIRED FOR RAPID REPAIR OF X-RAY-INDUCED DNA STRAND BREAKS IN VIVO.

A much higher yield of DNA single-strand breaks was obtained in the DNA polymerase-deficient mutant Escherichia coli K-12 pol A1 after a given dose of x-rays than had been found before in Escherichia coli. The increased yield of single-strand breaks was due to the absence of a rapid repair system, which had not been described in Escherichia coli K-12. This absence probably accounts for the x-ray sensitivity of the pol A1 mutant. The rapid repair system can be reversibly inhibited in pol+ cells.

Quantitation of the Involvement of the recA, recB, recC, recF, recJ, recN, lexA, radA, radB, uvrD, and umuC Genes in the Repair of X-Ray-Induced DNA Double-Strand Breaks in Escherichia coli

Isogenic Escherichia coli strains carrying single DNA-repair mutations were compared for their capacity for (i) the repair of X-ray-induced DNA double-strand breaks (DSB) as measured using neutral sucrose gradients; (ii) medium-dependent resistance, i.e., a recA-dependent X-ray survival phenomenon that correlates closely with the capacity for repairing DSB; and (iii) the growth medium-dependent, recA-dependent repair of X-ray-induced DNA single-strand breaks (SSB) as measured using alkaline sucrose gradients (about 80% of these SSB are actually parts of DSB). These three capacities were measured to quantitate more accurately the involvement of the various genes in the repair of DSB over a wide dose range. The mutations tested were grouped into five classes according to their effect on the repair of X-ray-induced DSB: (I) the recA, recB, recC, and lexA mutants were completely deficient; (II) the radB and recN mutants were about 90% deficient; (III) the recF and recJ mutants were about 70% deficient; (IV) the radA and uvrD mutants were about 30% deficient; and (V) the umuC mutant resembled the wild-type strains in its capacity for the repair of DSB.

Photochemical addition of amino acids to 14C-uracil

Abstract A survey was performed of the ability of the 22 common amino acids to add photochemically (2537A) to 14C-uracil. The 11 reactive amino acids were glycine, serine, phenylalanine, tyrosine, tryptophan, cystine, cysteine, methionine, histidine, arginine and lysine. The most reactive amino acids were phenylalanine, tyrosine and cysteine. The relevance of these reactions to the mechanism by which DNA and protein are cross-linked in vivo by ultraviolet light is discussed.

Physical and Chemical Changes Induced in Nucleic Acids by Ultraviolet Light

A number of different ultraviolet (UV)-induced alterations in DNA can now be measured chemically and physically. Currently it appears that we can explain at least some of the biological effects of UV irradiation in terms of specific chemical changes in the DNA. The purpose of this report is to summarize the different lesions produced in DNA, to assess if possible their biological importance, and to point up areas for future research. For a more comprehensive coverage of certain areas of the photochemistry of the nucleic acids, the recent reviews by Setlow (1), Shugar (2, 3), Smith (4), and Wacker (5) should be consulted.

INFLUENCE OF ULTRAFAST REPAIR PROCESSES (INDEPENDENT OF DNA POLYMERASE I) ON THE YIELD OF DNA SINGLE-STRAND BREAKS IN ESCHERICHIA COLI K-12 X-IRRADIATED IN THE PRESENCE OR ABSENCE OF OXYGEN.

The rate of production of DNA single-strand breaks has been studied in several polA1 derivatives of Escherichia coli K-12 x-irradiated in the presence or absence of oxygen. For log phase cells irradiated in phosphate-buffered saline, pH 7.3, the rate of production of breaks per single-strand genome per krad was 2.13 in the presence of air, and 0.66 in its absence [an oxygen-nitrogen breaks ratio (ONBR) of 3.2]. To determine if this oxygen effect was due to a difference in the absolute yield of breaks or to a differential ability for repair, the yield of single-strand breaks was studied in cells which had been inactivated either by heat treatment (52°C) or cold shock (0°C). In both cases there was a large increase in the yield of radiation-induced anoxic breaks (2.8-fold) and a small increase (1.25-fold) in aerobic breaks. Attempts were also made to inhibit repair using chemicals. Sodium cyanide (1 mM) used at 0°C had no effect on the anoxic yield of breaks. However, in the presence of quinacrine (0.2 mM) ...

MEMBRANE DAMAGE CAN BE A SIGNIFICANT FACTOR IN THE INACTIVATION OF ESCHERICHIA COLI BY NEAR‐ULTRAVIOLET RADIATION

Abstract A DNA repair competent strain of Escherichia coli K‐12 showed sensitivity to inorganic salts (at concentrations routinely used in minimal media) after irradiation with broad spectrum near–UV radiation, at fluences that caused little inactivation when plated on complex growth medium. This effect was not observed with cells that had been exposed to 254 nm radiation. This sensitivity to minimal medium was increased by increasing the salt concentration of the medium and by increasing the pH of the medium. This sensitivity was greatly increased by adding to the medium a low concentration of commercial glassware cleaning detergent that had no effect on unirradiated cells or far‐UV irradiated cells. These findings may explain the large variability often observed in near‐UV radiation survival data, and demonstrate that, at least on minimal medium plates, membrane damage contributes significantly towards cell killing. This phenomenon is largely oxygen dependent.