David A. Youngs

Single-strand breaks in the DNA of the uvrA and uvrB strains of Escherichia coli K-12 after ultraviolet irradiation.

Abstract— DNA single‐strand breaks were produced in uvrA and uvrB strains of E. coli K‐12 after UV (254 nm) irradiation. These breaks appear to be produced both directly by photochemical events, and by a temperature‐dependent process. Cyclobutane‐type pyrimidine dimers are probably not the photoproducts that lead to the temperature‐dependent breaks, since photoreactivation had no detectable effect on the final yield of breaks. The DNA strand breaks appear to be repairable by a process that requires DNA polymerase I and polynucleotide ligase, but not the recA, recB, recF, lexA101 or uvrD gene products. We hypothesize that these temperature‐dependent breaks occur either as a result of breakdown of a thermolabile photoproduct, or as the initial endonucleolytic event of a uvrA, uvrB‐independent excision repair process that acts on a UV photoproduct other than the cyclobutane‐type pyrimidine dimer.

R.B.E. of 50 kVp X-rays and 660 keV γ-rays (137Cs) with Respect to the Production of DNA Damage, Repair and Cell-killing in Escherichia Coli K-12

We have compared the efficiency of cell-killing, DNA single-strand breakage and double-strand breakage in an Escherichia coli K-12 wild-type strain after irradiation with soft X-rays (50kVp) and hard gamma-rays (600 keV) under aerobic conditions. Irradiation with 50 kVP x-rays resulted in 1.47 times more cell killing than was observed with 137Cs gamma-rays based on a comparison of D0 values evaluated from the survival curves. DNA sedimentation studies showed that, although 50 kVp X-rays were 1-93 times more effective than 137Cs gamma-rays in producing DNA double-strand breaks, there was no sigificant difference between the two qualities of radiation with respect to the initial number of single-strand breaks produced. When the cells were irradiated and allowed to repair maximally in minimal medium, 1-57 times more unrepaired DNA single-strand breaks remained per krad after irradiation with 50 kVp X-rays than with 137Cs gamma-rays. The increased yield of DNA double-strand breaks resulting from 50 kVp X-radiation may account for most of these additional unrepaired single-strand breaks, since single- and double-strand breaks are indistinguishable on alkaline sucrose gradients. These results suggest that the greater r.b.e. of 50 kVp X-rays may be related to an increased effectiveness for producing DNA double-strand breaks compared with the higher energy 137Cs gamma-rays.

The involvement of polynucleotide ligase in the repair of UV-induced DNA damage in Escherichia coli K-12 cells

SummaryThe effect of the ligts-7 mutation on cell survival and the extent of DNA repair after UV (254 nm) irradiation was determined for wild-type and uvrB5 cells of E. coli K-12 at 30° and 42°C. At the restrictive temperature (42°C) the ligts-7 mutation resulted in (i) a decrease in the extent of repair of DNA incision breaks arising during the excision repair process, and (ii) a decrease in the extent of post-replicational repair of gaps in newly-synthesized DNA. These deficiencies in DNA repair correlated with increases in cellular sensitivity to killing by UV radiation. Thus, DNA ligase plays an important role in vivo in both the excision and post-replicational repair processes.

SENSITIZATION OF ULTRAVIOLET‐IRRADIATED Escherichia coli K‐12 BY DIFFERENT AGARS: INHIBITION OF A rec AND exr GENE‐DEPENDENT BRANCH OF THE uvr GENE‐DEPENDENT EXCISION‐REPAIR PROCESS

Abstract—E. coli K‐12 wild‐type cells plated immediately after UV irradiation had a much lower survival on minimal medium solidified with agar‐agar No. 3 (Oxoid), purified agar (Difco) or ionagar (Colab) than on plates solidified with laboratory‐washed Noble agar (Difco). An intermediate survival was obtained on plates solidified with unwashed Noble agar (Difco). When irradiated cells were incubated in liquid minimal medium for various times before subsequent plating on agar‐agar No. 3, their survival increased rapidly and became identical to the survival of cells plated on washed Noble agar. The same phenomenon was found with polA 1 cells, but no differences in survival on the different agars were observed with uvrB, exrA, recA or recB cells. These‐ data suggest that a repair process dependent on the uvrB, recA, recB and exrA (but not the polA) gene products is inhibited when UV irradiated E. coli K‐12 cells are plated on minimal medium solidified with agar‐agar No. 3, purified agar or ionagar. This implies that the uvr gene‐dependent excision‐repair process consists of at least two branches, one controlled by the polA gene and a second controlled by the recA, recB and exrA genes and inhibitable by a substance present in certain agars. For maximum sensitivity, experiments designed to study chemical inhibitors of repair should use agar plates that do not themselves inhibit repair.

The Yield and Repair of X-Ray-induced Single-Strand Breaks in the DNA of Escherichia coli K-12 Cells

The yield of single-strand breaks present in chromosomal DNA of Escherichia coli cells after aerobic x irradiation was determined using alkaline sucrose gradient techniques. Experimental conditions were used that avoided a centrifugation speed dependence effect on DNA sedimentation, and allowed either minimal or complete enzymatic repair to occur. The results confirm the qualitative conclusions made in earlier reports from this laboratory, but indicate that the extent of DNA strand breakage was previously underestimated by factors of 3.4 to 7.6, depending on the strain and repair conditions used. Technical and interpretive difficulties in the measurement of DNA single-strand breakage using alkaline sucrose gradient techniques are discussed briefly. In the present experiments, the initial yield of DNA single-strand breaks was found to be 32.4/genome krad/sup -1/ (9.0 eV/break).

Influence of a UvrD Mutation on Survival and Repair of X-irradiated Escherichia Coli K-12 Cells

The presence of a uvrD mutation increased the X-ray sensitivities of E. coli wild-type and polA strains, but had no effect on the sensitivities of recA and recB strains, and little effect on a lexA strain. Incubation of irradiated cells in medium containing 2,4-dinitrophenol or chloramphenicol decreased the survival of wild-type and uvrD cells, but had no effect on the survival of recA, recB and lexA strains. Alkaline sucrose gradient sedimentation studies indicated that the uvrD strain is deficient in the growth-medium-dependent (Type III) repair of DNA single-strand breaks. These results indicate that the uvrD mutation inhibits certain rec+lex+-dependent repair processes, including the growth-medium-dependent (Type III) repair of X-ray-induced DNA single-strand breaks, but does not inhibit other rec+lex+-dependent processes that are sensitive to 2,4-dinitrophenol and chloramphenicol.

EXCISION REPAIR AND MUTAGENESIS ARE COMPLEX PROCESSES

ABSTRACT Excision repair in Escherichia coli has been divided into two major pathways: a growth medium-independent, polA+-dependent pathway, and a growth medium, recA+, recB+, lexA+, polC+, uvrD+-dependent pathway. The former pathway handles most of the UV radiation-induced lesions, and produces short patches of repair replication. The latter pathway can be subdivided into two branches: a uurD+-dependent branch, and a uvrD+-independent branch. Long patch repair replication appears to occur via this latter branch; it is recA+, lexA+ and poiB+-dependent, and is inhibited in buffer or by chloramphenicol (CAP). The uvrD+gene product can also function independently of recA+ if UV-irradiated cells are held in buffer for a time before plating. Thus, liquid holding recovery (LHR), an excision repair process that is observed in recA strains, is blocked by a uvrD mutation, but is enhanced by lexA and recB mutations. UV radiation-induced mutations are produced by excision repair as well as postreplication repair. In either case, they appear to occur via two subbranches of these repair systems; one controlled by recB+ and the other by uvrD+.

Involvement of uvrD, exrA, and recB Genes in the Control of the Postreplicational Repair Process

Ultraviolet radiation survival studies support the hypothesis that the uvrD, exrA and recB mutations inhibit separate branches of the postreplicational repair process.

Sensitivity to X-radiation of strains of Escherichia coli K-12 which lack DNA polymerase II

SummaryThe polB1 and polA1 polB1 strains of E. coli K-12, wihch are deficient in DNA polymerase II and in DNA polymerases I and II, respectively, were found to have essentially the same sensitivity to anoxic or aerobic X-irradiation as their related wild-type and polA1 strains, respectively. Thus, DNA polymerase II appears to play no major role in the repair of X-ray damage.