THE ROLE OF HOST-CELL REPAIR IN LIQUID-HOLDING RECOVERY OF U.V.-IRRADIATED ESCHERICHIA COLI

Abstract— The experiments reported give evidence that liquid-holding recovery (LHR) of u.v. irradiated E. coli cells involves basically the same type of dark repair which causes reactivation of phage and which results in much increased survival of the cells themselves [host-cell reactivation (HCR)]. LHR is very small in the two HCR(-) strains B syn^- and B_s-1, but occurs to larger but different extents in the three HCR(+) strains B, B/r, and B/r (Λ). LHR is inhibited if the liquid contains caffeine or acriflavine, both of which are known to inhibit HCR. The results indicate that most of the LHR effect, if not all, occurs during the liquid holding, rather than under growth conditions after liquid holding. It is assumed that the holding itself allows a prolonged time for, and therefore an enhancement of, HCR. It is thus implicit that LHR can be observed only where otherwise HCR of repairable u.v. damage would be incomplete, and that different extents of LHR, as observed in the three HCR(+) strains, reflect different extents of incompleteness of HCR. It is concluded that the repairable u.v. hits which are not fully repaired by HCR are predominantly those concerned with the extra u.v. sensitivity of the strains B and B/r (Λ), relative to B/r.     

REPAIR OF ULTRAVIOLET-DAMAGED TRANSFORMING DNA IN A MISMATCH REPAIR-DEFICIENT STRAIN OF HAEMOPHILUS INFLUENZAE

Abstract— Ultraviolet inactivation of Haemophilus influenzae transforming DNA followed inverse square root kinetics in both mismatch repair-proficient(hex₊) and deficient (hex-1) recipients. No DNA concentration effect was seen with UV-excision repair-deficient(uvr_-) strains. Low-efficiency genetic markers remained more sensitive than high-efficiency ones when they were assayed on excision repair-deficienthex₊ uvr_- strains. They were equally resistant whenhex₊ uvr_- recipients were used. We explain this by assuming that recombinational repair of UV lesions in the donor strand and mismatch repair of the recipient strand may overlap and cause double strand interruptions. This will eliminate low-efficiency transformants.     

ACTION SPECTRUM FOR THE OXYGEN-INDEPENDENT INACTIVATION OF HAEMOPHILUS INFLUENZAE TRANSFORMING DNA WITH NEAR ULTRA-VIOLET LIGHT*

Abstract— The oxygen-independent inactivation of Haemophilis influenzae transforming DNA by near UV light (300–380 nm) has an action spectrum in which the efficiency of inactivation drops rapidly between 313 and 334 nm and more slowly between 334 and 405 nm, with a shoulder between 334 and 365 nm.     

REVISED SPECTRA FOR THE INACTIVATION OF HAEMOPHILUS INFLUENZAE TRANSFORMING DNA BY MONOCHROMATIC ULTRAVIOLET LIGHT: EFFECT OF HISTIDINE

Abstract—It was reported previously that histidine sensitizes the genetic activity of Haemophilus influenzae transforming DNA to pure 334 nm ultraviolet light, Further measurements show that this apparent 334 nm sensitization was probably erroneous and that in fact, histidine protects DNA against inactivation by 334 nm light. This is now consistent with all previous observations that transforming DNA is protected by histidine against all near-UV wavelengths (above 320 nm) investigated.
A modified spectrum for the protection of H. influenza transforming DNA by histidine against ultraviolet light is described.     

NITROUS ACID REACTIVATION OF ULTRAVIOLET-IRRADIATED DENATURED TRANSFORMING DNA

Abstract— Partial recovery of ultraviolet-damaged denatured transforming DNA from Haemophilus influenzae , was obtained by exposing irradiated denatured DNA to nitrous acid and assaying transforming activity. This reactivation was affected by the time of incubation with nitrous acid.     

EFFECTS OF SENSITIZED AND UNSENSITIZED LONGWAVE U.V.-IRRADIATION ON THE SOLUTION PROPERTIES OF DNA

Abstract— Two types of photoreactions occur in DNA irradiated in aqueous systems with longwave u.v.-light (Λ > 295 nm), namely, (a) thymine dimerization, and (b) single- and double-strand breakage of the sugar phosphate backbone; these two reactions are unrelated. The presence of acetophenone as a photosensitizer caused an increase in dimerization by a factor of 16, and an increase in single-strand breaks by a factor of 4. The number of thymine dimers per single-strand break is about 100 in the sensitized and 25 in the unsensitized reaction. The alteration of the radius of gyration of DNA molecules is that expected by the degradation observed. At the same time the change in hyperchromicity is very small. Therefore as far as can be detected by these methods of investigation the gross conformation of the DNA double helix is stable against thymine dimerization.     

ON THE MECHANISM OF RADIOSENSITIZATION BY 5-BROMOURACIL. THE OCCURRENCE OF DNA STRAND BREAKS IN U.V.-IRRADIATED PHAGE T4 AS INFLUENCED BY CYSTEAMINE

Abstract— U.V.-irradiation of phage T 4Bo^r results in a decrease in sedimentation rate of BU-DNA which is attributed to single- and double-strand breaks. No breaks could be observed in unsubstituted DNA. Cysteamine present during u.v.-irradiation is able to prevent double-strand breaks but does not influence the production of single-strand breaks measured by alkaline sucrose gradient centrifugation. The biological importance and nature of DNA strand breaks due to BU-incorporation as well as the action of the protective agent on these breaks and on the biological activity are discussed.     

ULTRAVIOLET SENSITIVITY OF THE ADDITION, DELETION and REPLACEMENT OF LONG NONHOMOLOGOUS DNA SEGMENTS BY GENETIC TRANSFORMATION OF HAEMOPHILUS INFLUENZAE

Abstract— We have recently described the construction and some properties of Haemophilus influenzae Rd strains with long and different R plasmid-derived DNA segments (nonhomologous inserts) at the same site in the HP1 prophage. These inserts can be added to a recipient's genome by genetic transformation, they can be deleted from the recipient genome, or they can be replaced by another insert. We report here that the UV inactivation of all three phenomena followed single hit kinetics. Deletion was roughly 10 times more resistant; its UV-sensitivity equaled that of a high-efficiency point mutation. There was an inverse correlation between UV-sensitivity and additive transformation efficiency of the various inserts; sensitivity may thus be a measure of insert size. This correlation was not seen for deletion. All three phenomena were more sensitive when they were measured on excision repair-deficient uvr_- recipients. The dose-reduction factor for addition was about 1.5 while it was about 2.6 for deletion.     

COMPARISON OF SURVIVAL OF A U.V.-IRRADIATED URACIL-REQUIRING STRAIN OF ESCHERICHIA COLI B IN THE PRESENCE OF CHLORAMPHENICOL AND IN THE ABSENCE OF THE ESSENTIAL METABOLITE

Abstract— Restoration of viability of E. coli B U- after a total dose of 900 ergs/mm2 U.V. in the presence of chloramphenicol and in the absence of uracil was studied. Both treatments result in the recovery of the same fraction of the irradiated bacterial population. There is no additive effect if both treatments are applied to the same irradiated culture.     

DECREASED SURVIVAL RESULTING FROM LIQUID-HOLDING OF U.V.-IRRADIATED ESCHERICHIA COLI C AND SCHZZOSACCHAROMYCES POMBE

Abstract— Ultraviolet-irradiated cells of E. coli C and of haploid wild type yeast Schizosac-charomyces pombe , held in buffer at 22°-25°C for various periods of time prior to plating, show a lower survival than those plated immediately after irradiation. This 'negative liquid-holding effect' (NLHE) contrasts 'liquid-holding recovery' (LHR), found in a number of other E. coli strains and in Saccharomyces cerevisiae . NLHE was observed at all u.v. doses tested. The effect is maximal at holding temperatures in the range 25–30°C, it is very small at 5°C and (in E. coli C) at 44°C. NLHE and LHR resemble each other in several respects. In E. coli both effects are inhibitable by the dark repair inhibitors acriflavine, caffeine and potassium cyanide. They do not occur in nutrient broth, and they are much reduced if the irradiated cells were illuminated with photoreactivating light before holding. NLHE in S. pombe shows characteristics similar to those observed in E. coli C . Mutations leading to increased u.v. sensitivity in E. coli C and S. pombe can alter the liquid-holding response so that LHR is observed. Tetrad analysis of crosses between u.v.-sensitive and u.v.-resistant S. pombe strains indicates that a single chromosome region can control both u.v. sensitivity and liquid-holding response. Several possibilities explaining NLHE are discussed. From current knowledge about dark repair processes and from the similarities between NLHE and LHR in E. coli it seems likely that the two effects reflect slight changes in the efficiency of dark repair.     

THE ACTION OF U.V. LIGHT OF VARIOUS WAVELENGTHS ON PAPAIN

Abstract— Papain, prepared according to Kimmel and Smith is activated by irradiation with u.v. light of 254, 280 and 313 nm (φ_A= 0·022 ± 10 per cent). This activation is caused by the reductive splitting of a mixed disulfide in position 25. This disulfide is split with higher probability than any of the three structural disulfide links. This selectivity is likely caused by specific reduction of the mixed disulfide by thiol groups produced elsewhere in the molecule, although specific reduction by direct photochemical processes cannot be completely excluded on the basis of the data available. The quantum yields for destruction of structural cystine residues at the three wavelengths are comparable with the yields of cystine destruction in other proteins. The data also confirm that quanta absorbed by aromatic amino acid residues contribute to cystine destruction. In contrast to other enzymes with structural disulfide bonds, however, no correlation was found between the destruction of disulfide links and loss in activity. The results suggest that the mechanism of papain inactivation is not only dependent on the wavelength, but also on the dose.     

THE U.V. SENSITIVITY OF BACTERIA: ITS RELATION TO THE DNA REPLICATION CYCLE

Abstract— A striking increase in the shoulder of the u.v. survival curve but no change in the limiting slope is obtained when cultures of Escherichia coli strain TAU complete the DNA replication cycle in the absence of concommitant protein synthesis prior to irradiation. The u.v. sensitivity of protein synthesis or RNA synthesis is not altered significantly by this treatment.
In contrast to the result for strain TAU, there is no significant change in the u.v. survival curve for the u.v. sensitive E. coli B_s-1 when its DNA replication cycle is completed under similar conditions.
Following a period of inhibited protein synthesis there is a delay in the reinitiation of the normal DNA replication cycle when protein synthesis resumes. This delay would allow time for an intracellular repair system to operate before the attempted resumption of normal replication. Strain B_s-1, which is deficient in this repair system, would not be expected to benefit from such a delay, as consistent with the observed results. A model is presented to account for lethality due to attempted DNA replication during a period of repair synthesis. The maximum survival for a given u.v. dose would be predicted for a culture which has completed the normal DNA replication cycle prior to irradiation and which is not permitted to reinitiate the cycle until all possible repair synthesis is completed.     

THE EFFECT OF NATIVE ISOLOGOUS DNA ON THE INCORPORATION OF THYMIDINE-H3INTO U.V. IRRADIATED L-STRAIN CELLS

Abstract— The effect of highlypolymerized deoxyribonucleic acid (DNA) upon the incorporation rate of thymidine-H³ into DNA of recipient L -strain cells has been studied. The rate of incorporation of thymidine-H³ is much faster in controls and irradiated cells than in irradiated treated with isologous DNA.     

THE U.V. PHOTOCHEMISTRY OF CYTIDYLIC ACID

Abstract The ultraviolet (u.v.) irradiation of the 3' isomer of cytidylic acid (Cp) produces the hydrate (Cp*) with water added across the 5–6 double bond. The yield of this photo-product has been measured by, (a) separating the photoproduct by electrophoresis and (b) by observing the loss in absorbance. When corrections are made for reversal of the hydrate during the experiment, both methods gave the same result. Cross sections and quantum yields for the production of the hydrate were measured over the wavelength range 220 to 290 nm and over a pH range from 1 to 10. The quantum yield is markedly dependent on pH being higher by a factor of 6 to 10 for the neutral form. We have also demonstrated the existence of a very short lived photoproduct (half life 8–9 min) in both Cp3' and Cp5': The nature of this short lived product is not known.     

THE ROLE OF PEROXIDES IN THE INHIBITION OF DNA SYNTHESIS IN CELLS FOLLOWING IRRIDIATION WITH A U.V. MICROBEAM

Abstract Previous work had shown that the rate of DNA syntheses in mammalian cells could be reduced when a microbeam of ultraviolet light was directed into the medium surrounding the cells. The present work was designed to gather informtion on the mechanism of this effect.
Results show that catalase does protect the cellualar DNA synthesis under the following conditions.
(1) The microbeam must be directed into the surrounding medium—there is no protection if it strikes any part of the cell.
(2) The catalase must have been in contact with the cells for at least 4 hr before irradiation.
(3) At high closes of radiation the protection is only partial.
The catalase was labelled with fluorescent dye to demonstrate that it was able to penetrate the cell cytoplasm but not nucleus. Morphological studies on cell exposed to medium containing hydrogen pereoxide showed that protection was provided by intracellular and not extracellular catalasse and supported the idea that the toxic product produced by ultraviolet radiation of the medium was ultimately a peroxide.