Discontinuities in the DNA synthesized in an Excision-defective strain of Escherichia coli following ultraviolet irradiation

Abstract

Although Escherichia coli K12 uvrA6 is defective in the excision of pyrimidine dimers from its DNA, 37% of cells survive a dose of ultraviolet light which is equivalent to about 50 pyrimidine dimers per 107 nucleotides. The amount of tritiated thymidine incorporated into the DNA of irradiated cells indicates that pyrimidine dimers in the DNA inhibit DNA synthesis but are not permanent blocks. Zone sedimentation of single-strand DNA was performed in alkaline sucrose gradients. To minimize degradation by shearing, the DNA was released from spheroplasts layered on top of the gradients. Newly synthesized, denatured DNA from unirradiated cells sediments with a molecular weight of greater than 100 × 106, whereas the newly synthesized, denatured DNA from cells irradiated with 60 ergs/mm2 has a molecular weight of about 14 × 106. During subsequent incubation of the irradiated cells, the sedimentation rate of the DNA synthesized immediately after irradiation increases and approaches that of normal DNA. However, at any time during this incubation period, the incorporation of tritiated thymidine into fast-sedimenting DNA is minimal, suggesting that the daughter-strand DNA synthesized after ultraviolet-irradiation contains gaps, or alkalilabile bonds. These dicontinuities disappear during further incubation, as a higher rate of sedimentation is found. The number of these daughter-strand defects is similar to the number of pyrimidine dimers in an equivalent length of parental DNA.