Walter Harm

Mutants of phage T4 with increased sensitivity to ultraviolet.

Abstract After treatment of phage T4 with nitrous acid, three independent mutants with increased UV sensitivity have been isolated. The survival curves of two of them, both in darkness and under conditions of maximum photoreactivation, are very similar to those of phage T2. Genetic crosses have shown that the mutations had occurred in the v cistron (formerly called u cistron), which controls the differences in UV sensitivity and photoreactivability between T4 and T2; therefore, these two mutants are called v 1 and v 2 . The third mutation, producing a sensitivity level intermediate between T4 and T2, concerns a cistron called x which is not closely linked to v and affects UV sensitivity by a still unknown mechanism. In the recombinant v 1 x , the effects are additive, leading to a UV sensitivity considerably higher than that of T2. Thus, there are now available in phage T4 four sensitivity levels, represented by the genotypes v + x + (wild type), v + x , v x + , and v x , with relative sensitivities of 0.45, 0.75, 1.0, and 2.0, respectively. The slopes of the multicomplex survival curves obtained with the different sensitivity types do not show any simple correlation with the slopes of the monocomplex survival curves. Further, the multicomplex curves of the mutants have shoulders considerably lower than those of T4 and T2 wild type.

Differential effects of acriflavine and caffeine on various ultraviolet-irradiated Escherichia coli strains and T1 phage

Abstract Caffeine and acriflavine are known to inhibit repair of UV lesions in DNA of host-cell-reactivating [HCR(+)] Escherichia coli strains. Large differences were observed between various strains: B, B/r(λ) and K12(λ), carrying extra UV sensitivity relative to B/r and K12S, respectively, show much greater repair inhibition than the latter strains. This seems to be related to the (normally) rather incomplete repair in B, B/r(λ) and K12(λ), which was inferred from results on liquid-holding recovery 6 . Both incompleteness of repair and its great susceptibility to inhibition by caffeine or acriflavine could be explained by the assumption that UV lesions related to the extra sensitivity require the repair early . This assumption seems plausible at least for the extra sensitivity resulting from lysogenicity. Decreased UV survival in the presence of acriflavine or caffeine was also found in HCR(−) strains. B s−1 and B syn − , differing considerably in their UV sensitivities, show equal survival (at levels between 1 and 10 −4 ) if incubated with 7.5 ωg/ml acriflavine. The possible interpretation that all HCR(−) strains tested exhibit residual repair activity in the absence of acriflavine or caffeine, is supported by the finding that HCR(−) strains also show a slight residual HCR activity, inhibitable by acriflavine or caffeine, toward UV-irradiated phage Tr. However, part of the decreased cell survival might be due to synergistic effects between UV and acriflavine. At the highest applicable concentration, acriflavine is a stronger repair inhibitor than caffeine. However, neither substance decreases UV survival of HCR(+) cells or phage infecting HCR(+) cells to the level to which survival of HCR(−) cells or phage infecting HCR(−) cells is decreased by acriflavine or caffeine.

ANALYSIS OF PHOTOENZYMATIC REPAIR OF UV LESIONS IN DNA BY SINGLE LIGHT FLASHES. II. IN VIVO STUDIES WITH ESCHERICHIA COLI CELLS AND BACTERIOPHAGE.

Abstract Intracellular complexes of UV lesions in DNA with molecules of photoreactivating enzyme (PRE) in E. coli can be photolysed with a probability close to 1 by single light flashes of about 1 millisecond duration. The observed photoreactivation (PR) effect permits the number of PRE-substrate complexes present at the time of the flash to be determined on the following basis: ( a ) The number of PRE-substrate complexes equals the number of lesions repaired; ( b ) The number of photorepairable UV lesions present in DNA equals the number of pyrimidine dimers recoverable from acid hydrolysates; a UV dose of 1 erg·mm −2 of 2537 A radiation causes the formation of approximately 6.5 dimers per E. coli chromosome; ( c ) The observed PR effect can be expressed by the formal “dose-reduction”, i.e. by the reduction in the number of dimers. The kinetics of intracellular complex formation can be followed by varying the time interval between UV irradiation and flash reactivation. Stationary phase B s−1 cells, irradiated with 4.8 erg·mm −2 form a maximum number of complexes within approximately 5 min at room temperature, 50% of them being formed within the first 10–15 sec. For greater UV doses (6.4–24 erg·mm −2 ) the maximum number formed reaches a constant limiting value of 20, indicating that this is approximately the number of PRE molecules in these cells. Experiments with sequential flashes at various temperatures between 2° and 44° show that both the maximum extent of complex formation and the photolytic rate constant are temperature-independent in this range. Hence, the usually observed temperature-dependence of PR under conditions of continuous illumination reflects the temperature-dependence of the complex formation only. Extensive PR effects with single light flashes were also found in UV-irradiated phage T1 after injection of unirradiated B s−1 cells. The effects are much smaller in irradiated host cells due to competition by the bacterial DNA. Creating the competitive substrate after the phage DNA has reacted with the PRE results in a time-dependent decrease of the PR effect in phage as the enzyme equilibrates between the host-cell and phage DNAs. The slow rate of equilibration indicates a relatively high stability of the complexes in the dark. A heterogeneity of the photoproducts is evident with regard to both complex formation and stability. Comparative experiments with phage infecting other host strains indicate that the number of PRE molecules in strain B/r equals that in B s−1 , but is lower in the K12 derivatives AB 1157, AB 2437 and AB 2480.

On the relationship between host-cell reactivation and UV-reactivation in UV-inactivated phages.

SummaryHost-cell reactivation (HCR) and UV-reactivation (UVR) were studied in phage T1, T3 and λ, using as host bacteriaE. coli B, C, andK12S, as well as their non-hostreactivating mutantsBs−1 (Ellisonet al. 1960),C syn−(Rörschet al. 1962), andK12S hcr−. The experiments gave further support to the idea that HCR is an enzymatic process. It repairs about 80 to 90 percent of otherwise lethal UV-lesions not only in phage DNA, but also in bacterial DNA. Thehcr− mutant isolated fromK12S for the purpose of this investigation, and thesyn− mutant of ColiC show a very small extent of HCR; they are not completely deficient for the HCR-enzyme.A correlation exists between the occurrence of HCR and UVR. UVR is absent in those cases where no HCR is observed. In systems with residual HCR-activity (hcr− andsyn− cells) UVR is less pronounced and has its maximum at lower UV-doses than in systems with full HCR-activity. UVR occurs also in unirradiated host-reactivating cells, if a large number of additional UV-lesions is introduced by means of superinfecting homologous phage. This effect is not observed in non-hostreactivating strains. The hypothesis is discussed that UVR is not a specific repair phenomenon by itself, but is the result of inhibition of cellular processes tending to decrease the survival.

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 Bs‐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.

EFFECTS OF DOSE FRACTIONATION ON ULTRAVIOLET SURVIVAL OF ESCHERICHIA COLI

Abstract— Exposure of E. coli B/r and B at low average dose rates of u.v. radiation (2537 Å), produced either by fractionated doses or by continuous irradiation at a very low dose rate (80 ergs/mm2/hr), results in much increased survival compared to single exposure at high dose rate. This increase is attributed to repair taking place during the irradiation period. The effect is small in the repair‐deficient strains E. coli B8‐1_, and C syn‐, and is absent in phage T1 and T4, which cannot undergo repair in the extracellular state. However, the prolonged time available for repair in these experiments accounts for only a very minor part of the increase in survival. The principal factor apparently is that the number of lesions present at any time remains relatively low. Presumably complete repair, not only the excision step, can occur in buffer during the irradiation period. This interpretation is supported by experiments in which cells were exposed to combinations of highly fractionated irradiation and single‐dose irradiation. We therefore propose that mutual interference in repair, possibly by overlapping of repair regions in complementary DNA strands, reduces considerably the repair efficiency if many lesions are present. This hypothesis explains the ‘shouldered’ survival curves of B/r and possibly other E. coli strains as due to decreasing repair efficiency with increasing u.v. dose

On the control of UV-sensitivity of phage T4 by the gene x

Abstract DNA of UV-irradiated phages T4 v + x + , T4 v 1 x + , T4 v + x , and T4 v 1 x was used for competitive inhibition of photo-enzymatic repair (photoreactivation) in vitro of UV-irradiated transforming DNA of Haemophilus influenzae . At a given UV-dose and at a given DNA concentration, the competitive power of all four DNA species was identical, indicating the presence of equal numbers of photorepairable UV-lesions in all cases. It is concluded that the quality and quantity of the primary UV-effects are identical for these four phage types, although they exhibit considerable differences in UV-sensitivity and in the extent of photoreactivation. Hence, not only the v gene, but also the x gene, controls the UV-sensitivity of phage by intracellular alteration of the UV-irradiated DNA. This conclusion is consistent with the results of rII + marker rescue experiments, showing that the extent of rescue is always lower if the unirradiated parental phages carry the x rather than the x + allele. A plausible explanation for these results is the control by the x gene of the frequency of genetic recombination, which is shown independently in crosses between unirradiated rII phages. To explain the increased UV-sensitivity of single-infecting T4 x phage, it is tentatively assumed that the x allele, in contrast to the x + allele, does not permit a repair process which has some mechanism in common with genetic recombination. An alternative explanation would be that the x mutation concerns more than one gene. Additional experiments were carried out to study the superposition of the x gene effect with v gene repair and photo-enzymatic repair in marker rescue.

Dark repair of photorepairable uv lesions in escherichia coli

Abstract Photoenzymatic repair (PER) immediately after UV irradiation of E. coli B/r abolishes approximately 67% of those UV lesions that usually do not become dark repaired (PR sector = 0.67). If the irradiated cells are held in bufer before undergoing PER, the PR sector decreases and becomes virtually zero after 24–48 h holding. Such an effect is essentially absent in the E. Coli strains Bs−1, AB 2437, and AB 2480 which are deficient in excision-resynthesis dark repair (ERR). In B/r, the decrease in the PR sector is much inhibited in the presence of potassium cyanide, caffein, and acriflavine. These and other results suggests that the observed loss of photoreactivability by holding in buffer is due to excision of photorepairable lesions. However, the fact that the decrease in PR sector is the result not only of an increase in dark survival (“liquid-holding recovery”), but also of a decrease in the absolute level of PR survival indicates that excision does not always lead to dark repair. Loss of photoreactivability during holding is only partial in strain AB 2463 (rec−, which is deficient in another type of dark repair (“REC repair”), and in E. coli B. No PR is observed in B/r cells UV-irradiated at an extremely low dose-rate (where they are known to undergo most extensive dark repair), while under the same conditions a reduced amount of PR is still found in AB 2463 (rec−) cells. Altogether, the results indicate that (1) in E. coli B/r under usual irradiation conditions PER repairs preferentially UV lesions that would otherwise be dark-repaired; (2) in fact, all of the photo-repairable lesions in E. coli are dark-repairable; and (3) complete dark repair of photo-repairable lesions in B/r requires both REC repair and a higher-than-normal extent of ERR.

Host-cell reactivation of non-lethal ultraviolet-effects.

Abstract— Delay of intracellular growth of u.v.‐irradiated bacteriophage T1 and Λ was compared in host‐cell reactivating [HCR(+)] and non‐host‐cell reactivating [HCR(—)] bacterial strains. At a given phage survival level, intracellular growth delay occurs to the same extent in HCR (+) and HCR (‐) strains; at a given absolute u.v.‐dose, this delay is considerably more expressed in HCR (‐) than in HCR (+) strains. Therefore, it does not reflect the time required for the HCR repair of otherwise lethal U.V. lesions. The results rather suggest that U.V. causes, besides lethal lesions, stable photoproducts in the DNA, which are a priori non‐lethal, and which are recognized and efficiently eliminated by the HCR repair system. The HCR enzymes likewise act on (non‐lethal) u.v.‐photoproducts causing prophage induction in lysogenic cells. Consequently, one obtains the maximum induction effect in a lysogenic HCR (‐) strain at a much lower u.v.‐dose than in the corresponding lysogenic HCR (+) strain. In contrast, u.v.‐damage causing loss of the host cells capacity to support growth of unirradiated phage is not affected by HCR.

Vergleichende Untersuchungen an HNO2-Inaktivierten und UV-Inaktivierten Bakteriophagen T4

SummaryThe inactivation of phage T4 by nitrous acid (HNO2) is essentially an exponential function of time of treatment. HNO2-inactivated T4 is able to undergo multiplicity reactivation, and genetic markers may be rescued by live phage, however, the extent of both effects is appreciably less than after UV-inactivation. Also, the survival of phenotypic function of the cistronsr II-A andr II-B is lower with HNO2-treatment than with a UV-irradiation of a corresponding number of hits.The reduced effects are quantitatively accounted for by the assumption of lethal hits blocking early steps of infection. These early-step damages amount to approximately 1/6 of the total hit number; it is still unknown whether they occur in DNA or in protein. Some indication for the occurrence in protein comes from the result that the host-killing efficiency of HNO2-inactivated phage is reduced at a similar rate as these early-step damages occur. However, at least 5/6 of the lethal hits are due to chemical changes within the DNA, as can be calculated from the results of multiplicity reactivation, marker rescue, and phenotypic survival of therII-cistrons.