Bryn A. Bridges

Mutagenesis in Escherichia coli

SummaryBoth thymine starvation and gamma radiation, like ultraviolet light, produce base change mutations to prototrophy in Escherichia coli and the Exr+ phenotype is involved in the mutagenic process. DNA strand breakage is a direct or indirect consequence of all three treatments suggesting that the filling of gaps in DNA by a process involving the exr gene product may be a common step in mutagenesis.

The Sensitivity of Escherichia Coli to Ionizing Particles of Different LETs

SummaryMeasurements have been made of the inactivation doses for Escherichia coli exposed to α-particles and protons of different LETs and also to γ-rays. These have been interpreted by target theory in terms of two types of lethal damage to the bacterial genome. Damage of type 1 affects only one strand of the DNA macromolecule and is partially reparable in vivo. Damage of type 2 following one track intersection of the DNA affects both strands and is irreparable. The identity of type 1 damage is uncertain; it does not appear to cause appreciable inactivation by producing ‘base’ mutations but may lead to a lesion recognizable in vitro as a single-strand break. Type 2 damage probably corresponds to double strand ‘scission’ of DNA as observed in vitro.

NON-PHOTOREACTIVATING REPAIR OF MUTATIONAL LESIONS INDUCED BY ULTRAVIOLET AND IONIZING RADIATIONS IN ESCHERICHIA COLI.

Abstract The action of one established and two presumed repair systems on premutational lesions induced by ionizing and ultraviolet radiations has been studied int he tryptophan auxotrops Escherichia coli B/r WP2 and E. coli WP2 hcr t− (which has a reduced ability to excise thymine dimers). Premutational lesions induced by ionizing radiation are not susceptible to excision-repair nor to the mutation-frequency decline which occurs with UV lesions in minimal media, but are removed by a slow-acting process at 16°. Premutational lesions induced by UV are removed by excision-repair, by mutation-frequency decline both at 37° in minimal medium and at 16° even on plates enriched with nutrient broth, and by a similar low-temperature process to that which acts at 16° on lesions induced by ionizing radiation. There is some evidence that mutation-frequency decline may be related to excision-repair, for example by the involvement of a common enzyme. Alternatively mutation-frequency decline could merely reflect the excision-repair of premutational lesions which are more amenable to excision than thymine dimers. The slow acting process at 16° appears to be quite distinct from excision-repair and mutation-frequency decline since, unlike these, it occurs normally in WP2 hcr − .

Ultraviolet Damage to Bacteria and Bacteriophage at Low Temperatures.

The survival of Escherichia coli B/r WP2 (tryptophan-requiring) from ultraviolet irradiation when suspended in 0.067M phosphate buffer (pH 7) has been studied over the temperature range 22� to -269�C. In unfrozen suspensions there was no appreciable change in sensitivity between 22� and -10�C. The sensitivity in the presence of ice progressively increased by a factor of 7 when the temperature was lowered to -79�C. Between -79� and -196�C the sensitivity decreased to less than four times the sensitivity at 22�C and was not appreciably different at -269�C. Evidence from experiments with bacteriophage T1 and E. coli WP2 HCR- (a strain unable to excise thymine dimers) indicates that a new, qualitatively different lesion, less amenable to repair, may replace the thymine dimer in E. coli irradiated at -79�C.

A NOTE ON THE MECHANISM OF UV MUTAGENESIS IN ESCHERICHIA COLI

Abstract Recent results on UV-induced reversion to prototrophy in Escherichia coli have been incorporated in a hypothesis in which it is postulated; ( 1 ) that two distinct UV-induced lesions in DNA are involved in the production of mutations, say events A and B; ( 2 ) that event A is a type of lesion known as thymine dimer-type damage which can be removed by photoreactivation (requiring light) or by the excision-repair (“host-cell reactivation”) system; ( 3 ) that event B is rapidly removed (“mutation-frequency decline”) by a repair enzyme which needs an energy source but not a nitrogen source and which is inhibited by amino acids or concomitant RNA and protein synthesis. Even B is also probably excisable by the host-cell reactivation system but is not monomerizable by the photoreactivation enzyme; ( 4 ) that events of type A act in mutagenesis by inhibiting the “mutation-frequency decline” enzyme and thus increasing the number of fixed mutations all of which arise from events of type B.

SUSCEPTIBILITY OF MILD THERMAL AND OF IONIZING RADIATION DAMAGE TO THE SAME RECOVERY MECHANISMS IN ESCHERICHIA COLI.

Abstract A correlation exists in E. coli between sensitivity to ionizing radiation and to thermal stress at 52°C. It is suggested that systems involved in recovery from damage inflicted by ionizing radiation are those which in a natural environment repair or bypass mild thermal damage, for example the breakage of single strands of the DNA duplex.

On the Nature of the Lethal and Mutagenic Action of Ultraviolet Light on Frozen Bacteria

The supersensitivity of bacteria to ultraviolet light (u. v. ) when irradiated at low temperatures in frozen suspension has been studied in relation to known cellular mechanisms for repair of radiation damage. Strains of Escherichia coli deficient in such mechanisms, besides being more sensitive to the lethal action of u. v., showed little or no additional supersensitivity at — 79 °C. The deficiencies studied were in (a) her locus controlling ability to excise pyrimidine dimers from DNA; (b) fil locus, probably acting at least in part by permitting more effective use of the excision-repair system; (c) rec locus, controlling an aspect of the ability to undergo recombination, presumably by action on DNA , also conferring X -ray resistance; (d) exr locus, controlling ability to repair X -ray damage, possibly by rejoining single-strand breaks. It is concluded that damage induced by u. v. at — 79 °C differs from that induced at 22 °C in being much less amenable to repair by the systems acting on DNA than damage induced in the non-frozen state. Supersensitivity of the reactivation systems themselves at — 79 °C was excluded (unless the reactivation system and the DNA are both inactivated by the same absorption event). Mutational damage induced by u. v. at — 79 °C differed from that induced at 22 °C in that the observed mutation frequency was less dependent upon immediate post-irradiation protein synthesis. Furthermore, such damage was not involved in the cumulative (‘dose-squared’) response when bacteria were thawed and irradiated further at 22 °C. The observation of Smith (see references) that the cross-linking ofDNA with protein is greater when exposure to u. v. is carried out at — 79 °C than at room temperature has been confirmed.

TEMPERATURE, TIME AND X-RAY MUTAGENESIS IN ESCHERICHIA COLI

Abstract Two effects of temperature in the range 16° to 37° on the induction by X-radiation of reversions to prototrophy in tryptophan-requiring Escherichia coli WP2 are described. 1. 1. The average number of mutable units which segregate from a nucleus at 16° (2.23) is greater than that at 37° (1.28). For this effect the temperature before irradiation is important and the most reasonable explanation postulates the existence at 16° of an appreciable G 2 period in the cell cycle during which DNA synthesis does not occur, so that in most nuclei two unseparated replicas of the genome are present. At 37° where it is known that any non-synthesizing period is very short, all nuclei presumably divide as soon as the chromosome has completed its replication. The segregation pattern at 37° is independent of whether irradiation is carried out anoxically or aerobically and implies that most if not all mutations are carried on both strands of the segregating DNA. 2. 2. At 16° fewer mutations are scored per unit dose than at 37°. This low temperature mutations loss is mainly dependent on the post-irradiation temperature. Unlike some other supposed restoration systems it is magnified rather than prevented by acriflavine. It has the characteristics of a metabolic process, but there is no evidence to show whether this process leads to death or to return to normality of the premutational lesion.

ULTRAVIOLET MUTAGENESIS IN ESCHERICHIA COLI AT LOW TEMPERATURES

Abstract The sensitivity of Escherichia coli B/r WP2 (tryptophan-requiring) suspended in phosphate buffer to the lethal and mutagenic action of UV light is reported over the temperature range 22° to −196°. In the unfrozen state the sensitivity is unchanged from 22° to −10°. In the frozen state the sensitivity relative to the unfrozen state increases by a factor of seven as the temperature is lowered to −79°. At −196° the factor is reduced to four. The increase in sensitivity is dependent upon the frozen state being maintained during irradiation. Experiments with a sensitive strain E. coli WP2 hcr − which has a reduced ability to excise thymine dimers reveal a complicated lethal response. At 22° the survival curve has a pronounced shoulder. At −79° this shoulder is abolished and the survival curve appears to run parallel to the ultimate slope of the curve at 22°. E. coli WP2 hcr − is 2.5 times more sensitive to the mutagenic action of UV at −79° than at 22°. No temperature dependence between −5° and −79° comparable with that observed with bacteria was observed in the destruction of thymine by UV in a system in vitro . In both systems, however, the sensitivity at −196° was less than that at −79°. The enhancement of UV damage in the frozen state is also discussed in terms of its possible significance in the evolution of species.

MUTAGENESIS IN ESCHERICHIA COLI. V. ATTEMPTED INTERCONVERSION OF OCHRE AND AMBER SUPPRESSORS AND MUTATIONAL INSTABILITY DUE TO AN OCHRE SUPPRESSOR.

SummaryA possible quantitative system for the interconversion of ochre and amber suppressors was studied in Escherichia coli WU36-10, a strain in which a leucine requirement is suppressed by amber suppressors and a tyrosine requirement is suppressed by ochre suppressors. The conversion of am Sup-2+ to oc Sup-2+ occurred at rates similar to those for the de novo induction of such suppressors, both spontaneously and after ultraviolet or gamma irradiation. Both induction and conversion of suppressors showed the phenomenon of “mutation frequency decline” after ultraviolet light. Conversions in the opposite direction from oc Sup-2+ to am Sup-2+ were, however, not detected in unmutagenised populations of oc Sup-2+ strains derived either by conversion from an am Sup-2+ strain or de novo from the parental WU36-10, nor were they detected after treatment with ultraviolet light, gamma radiation or 2-aminopurine. If the conversion of oc Sup-2+ to am Sup-2+ occurs at all, it is at a rate very considerably lower than that for the conversion of am Sup-2+ to oc Sup-2+. Some Tyr+ oc Sup-2+ mutants demonstrated mutation rates c. 100 times greater than those of WU36-10 for mutation to Leu+ spontaneously and after ultraviolet or gamma radiation. Possible explanations of this are discussed.