Indirect mutagenesis in phage lambda by ultraviolet preirradiation of host bacteria

The changes in molecular weight of deoxyribonucleic acid (DNA) synthesized after ultraviolte irradiation of Escherichia coli WP28 uvrA, and strains additionally mutant at polA, exrA, recA, and exrA and polA loci, were examined by alkaline sucrose gradient centrifugation. In a repari=deficient uvrA recA strain, the frequency of breaks in newly synthesized DNA was equal to that for pyrimidine dimers in parental DNA. Measurements of the amounts and rates of postreplication repair of these breaks indicate that (i) repair is two to three times faster when DNA polymerase I is present, although (ii) almost all breaks are repaired regardless of DNA polymerase I activity. (iii) Increased ultraviolet doses lead to an increase in the proportion of breaks remaining unrepaired in uvrA recA, UVRA exrA, and uvrA exrA polA strains. The numbers of unrepaired breaks resemble the numbers expected if repair of one lesion is prevented by proximity of a second lesion.

Genetic and kinetic evidence for different types of postreplication repair in Escherichia coli B.

— An action spectrum was obtained for photoreactivation of killing (PR) of Streptomyces griseus conidia. This spectrum shows a major peak around 436 nm, originally observed by A. Kelner, and a secondary peak at 313 nm not previously reported. The rate of PR shows a strong dependence upon temperature and dose rate of the PR light at 436 nm, but this decreases to only a slight dependence upon these parameters at 313 nm. These findings suggested that PR at 436 nm in this organism is of the usual photoenzymatic type, but that PR at 313 nm might be of a different kind.



A mutant (PHR-1) of S. griseus was found that shows only a narrow range of PR (roughly 310–400 nm) with a single peak at 313 nm. The PR efficiency was lower than for wild type and the PR sector not greater than one-half that of wild type. This PR shows no temperature dependence. Essentially similar behavior was observed with wild-type Streptomyces coelicolor. These findings show that at least some of the PR at 313 nm is a separable phenomenon. It is therefore unlikely to involve a mechanism identical to that at 436 nm.



The nature of PR at 313 nm in Streptomyces is not known. If it is enzymatic, it is remarkable in having little or no dependence upon temperature and dose rate. Absence of photoprotection and liquid-holding recovery indicate that it is not indirect PR. Some of it (that part exhibited by S. griseus PHR-1 and S. coelicolor) might result from a direct photochemical action on DNA.

Photoreactivation of killing in Streptomyces: action spectra and kinetic studies.

Deletion mutations (resistant to colicin B) were compared in Escherichia coli B strains that differ in DNA repair capacity; parental (with a B/r-type repair capacity), uvrA - or uvrB - (unable to excise pyrimidine dimers), pol - (defective in DNA polymerase I activity) and recA - (defective in recombinational repair). Strains uvrA - and uvrB - showed about 20 times greater sensitivity to deletion mutations as well as to killing by UV compared with the parental strain. However, they showed almost the same sensitivity to deletion mutations as well as to killing by X-rays compared with the parental one. A recA - strain showed no significant increase in the deletion yield above the spontaneous level after UV or X irradiation. Two pol - strains showed significantly higher deletion mutability to UV and X-rays than the parental strain. The rate (/cell division/bacterium) of spontaneous deletions was significantly higher in the two pol - strains than in the parental, uvrA -, uvcrB - and recA - strains, whereas spontaneous rates among the latter strains showed no significant difference. The Col-B R mutations were made of “short” (from colB to tonB locus) and “long” (from colB to trp locus) delections. Almost all the induced mutations were “short” deletions, but spontaneous ones were made of “short” or “long” deletions in about a one-to-one ratio except for the pol -strains, which had a short-to-long ratio of about 4:1. It is concluded that induction of deletion mutations by UV or X-rays requires not only pyrimidine dimers or X-ray damage to DNA but also participation of the recombination mechanism, and that the lack of DNA polymerase I activity may result in enhanced DNA errors which in turn lead to elevated deletion mutability.

Spontaneous and radiation-induced deletion mutations in Escherichia coli strains with different DNA repair capacities

An Escherichia coli strain carrying the recB21 and res-1 mutations showed an abnormally low level of colony-forming ability although it grew essentially normally in liquid medium. The recB21 res-1 strain showed little, if any, of the ultraviolet (UV)-induced deoxyribonucleic acid (DNA) breakdown characteristic of the res-1 mutant. Nevertheless, the double mutant was far more sensitive to UV than either the res-1 or the recB21 strain. When compared with a wild-type strain, the rate of release of dimers from UV-irradiated DNA was very slow in the recB21 res-1, but normal in the res-1 recB(+) or recB21 res(+) mutants. However, the ratio of dimer-to-thymine released into the acid-soluble fraction was three times higher than the wild type in recB21 res(+) and recB21 res-1 and only one-tenth as high as the wild type in res-1 rec(+). Alkaline sucrose gradient centrifugation revealed occurrence of single-strand incision of UV-irradiated DNA and the restitution of nicked DNA at a similar rate in the recB21 res-1 and recB21 res(+) strains. Mutants uvrC(-) showed increased amounts of nicks in their DNA with increasing incubation time after UV irradiation, although no detectable amounts of dimers were excised from UV-irradiated DNA. From these results, it is concluded that the increased sensitivity of the res-1 strain to UV light is due to a reduced ability to excise dimers from UV-irradiated DNA and that the high rate of UV-induced breakdown of DNA is not the primary cause. A possible role of uvrC gene in the excision repair is discussed.

Excision Repair Characteristics of recB−res− and uvrC− Strains of Escherichia coli

— Ultraviolet-sensitive strains of E. coli, Hs30 (uvr- phr- arg-), lacking the ability to repair ultraviolet damage in the dark (uvr-), lacking photoreactivating-enzyme activity (phr-), and lacking the ability to synthesize arginine (arg-), and Hs30–R (uvr- phr+ arg-), having the same auxotrophic marker and the same high u.v.-sensitivity, were derived, respectively, from resistant strain H/r30 (uvr+ phr- urg-) and its revertant H/r30–R (uvr+ phr+ urg-). Hs30 and Hs30–R have about 25 and 35 times higher sensitivity than H/r30 and H/r30–R for u.v.-induced killing and mutation to prototrophy, respectively. Efficiency of photoreactivation (PR) of mutation in strain Hs30–R is about ten times higher than the PR efficiency in the parent strain H/r30–R at wavelengths between 3341 and 4358 A but not that much higher at 3132 A. On the other hand, Hs30 shows no PR at any of these wavelengths, although the parent strain H/r30 does show PR of mutation around 3341 A., These results support the. previously described model that PR in H/r30 is an indirect effect of PR light which enhances dark repair. It is concluded that Hs30 and Hs30–R lack dark-repair capacity for u.v.-induced mutational damage and that the same type of photoreactivable DNA lesions as those responsible for U.V. killing, i.e. pyrimidine dimers, are the primary cause of u.v.-induced mutation in the uvr-strains. PR spectra for killing in Hs30–R and mutation in H/r30–R and H/r30 were obtained and compared, and the quantitative conclusions reached are discussed in connection with the hypothesis that the same kind of lesion (pyrimidine dimer) is the primary cause of mutation in the uvr+ strains as well.

Action spectra for photoreactivation of killing and mutation to prototrophy in U.V.-sensitive strains of Escherichia Coli possessing and lacking photoreactivating enzyme.

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Action spectra for photoreactivation of killing and mutation to prototrophy in U.V.-sensitive strains of Escherichia Coli possessing and lacking photoreactivating enzyme.