Rate zonal density gradient ultracentrifugation analysis of repair of radiation damage to the folded chromosome of Escherichia coli
Ulmer, Kevin M.
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The structure of the membrane-free nucleoid of Escherichia coli and of unfolded chromosomal DNA was investigated by sedimentation on neutral sucrose gradients after irradiation with 60Co gamma-rays and ultraviolet light (2S4nm). Irradiation both in vivo and in vitro was used as a molecular probe of the constraints on DNA~packaging in the bacterial chromosome. The extremely gentle lysis and unfolding procedures which were developed yielded undamaged, replicating genomes, thus permitting direct measurement of the formation and repair of DNA double-strand breaks at biologically-significant doses of ionizing radiation. In vitro UV-irradiation of nucleoids resulted in an increase in the observed rate of sedimentation due to the formation of an unknown photo-product. In contrast, UV-irradiation of wild-type cells in vivo showed evidence of the formation of incision breaks which resulted in the relaxation of supercoiling in the nucleoid. Strand breakage was also observed following in vivo UV-irradiation of a uvrB-5 strain, but at a lower rate and also accompanied by considerable unfolding of the chromosome. Such lesions may have been the result of direct photochemical reactions in the nucleoid, or enzyme activity associated with a uvr-independent mode of repair. The number of domains of supercoiling was estimated at 170 per genome equivalent of DNA based on measurements of relaxation caused by single-strand break formation in in vivo- and in vitro-gamma-irradiated folded chromosomes. Similar estimates based on the target size of RNA molecules responsible for maintaining the compact packaging of the nucleoid predicted negligible unfolding due to the formation of RNA single-strand breaks at doses up-to 10 Krad, and were born out by experimental measurements. Unfolding of the nucleoid in vitro by limit-digestion with RNase or by heating at 70° resulted in DNA complexes with sedimentation coefficients of 1030±59S and 625±15S respectively. The difference in these rates was apparently due to more complete deproteinization and thus less mass in the heated material. These structures are believed to represent intact, replicating genomes in the form of complex-theta structures containing 2-3 genome equivalents of DNA. The rate of formation of double-strand breaks was determined from molecular weight measurements of thermally unfolded chromosomal DNA gamma-irradiated in vitro. Break formation was linear with dose up to 10 Krad, resulting in 0.27 double-strand breaks per kilorad per genome equivalent of DNA and requiring 1080 eV/double-strand break. The influence of possible non-linear DNA conformations of these calculations is discussed. Repair of ionizing radiation damage to folded chromosomes was observed within 2-3 hours of post-irradiation incubation in growth medium. A model based on recombinational repair is proposed to explain the formation of 2200-2300S material during early stages of incubation and subsequent changes in the gradient profiles. Such behavior is not observed for post-irradiation incubation of wild-type cells in buffer or for a recA-13 strain incubated in growth medium. Association of unrepaired DNA with plasma membrane is proposed to explain the formation of a peak of rapidly sedimenting material (>>3100S) during the later stages of repair. Direct evidence of repair of double-strand breaks during post-irradiation incubation in growth medium was obtained from gradient profiles of DNA from RNAse-digested chromosomes. The sedimentation coefficient of broken molecules was restored to the value of unirradiated DNA after 2-3 hours of incubation, and the fraction of the DNA repaired in this fashion was equal to the fraction of cells which survived at the same dose. An average of 2.7 double-strand breaks per genome per lethal event was observed, suggesting that 1-2 double-strand breaks per genome are repairable in this strain of E. coli.
Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution April 1978
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