Rate zonal density gradient ultracentrifugation analysis of repair of radiation damage to the folded chromosome of Escherichia coli

The structure of the membrane-free nucleoid of Escherichia coli andof unfolded chromosomal DNA was investigated by sedimentation on neutralsucrose gradients after irradiation with 60Co gamma-rays and ultravioletlight (2S4nm). Irradiation both in vivo and in vitro was used as amolecular probe of the constraints on DNA~packaging in the bacterialchromosome. The extremely gentle lysis and unfolding procedures whichwere developed yielded undamaged, replicating genomes, thus permittingdirect measurement of the formation and repair of DNA double-strand breaksat biologically-significant doses of ionizing radiation.In vitro UV-irradiation of nucleoids resulted in an increase in theobserved rate of sedimentation due to the formation of an unknown photo-product.In contrast, UV-irradiation of wild-type cells in vivo showedevidence of the formation of incision breaks which resulted in the relaxationof supercoiling in the nucleoid. Strand breakage was also observedfollowing in vivo UV-irradiation of a uvrB-5 strain, but at a lower rateand also accompanied by considerable unfolding of the chromosome. Suchlesions may have been the result of direct photochemical reactions in thenucleoid, or enzyme activity associated with a uvr-independent mode ofrepair.The number of domains of supercoiling was estimated at 170 pergenome equivalent of DNA based on measurements of relaxation caused bysingle-strand break formation in in vivo- and in vitro-gamma-irradiatedfolded chromosomes. Similar estimates based on the target size of RNAmolecules responsible for maintaining the compact packaging of thenucleoid predicted negligible unfolding due to the formation of RNA single-strandbreaks at doses up-to 10 Krad, and were born out by experimentalmeasurements.Unfolding of the nucleoid in vitro by limit-digestion with RNase orby heating at 70° resulted in DNA complexes with sedimentation coefficientsof 1030±59S and 625±15S respectively. The difference in theserates was apparently due to more complete deproteinization and thus lessmass in the heated material. These structures are believed to representintact, replicating genomes in the form of complex-theta structurescontaining 2-3 genome equivalents of DNA.The rate of formation of double-strand breaks was determined frommolecular weight measurements of thermally unfolded chromosomal DNA gamma-irradiatedin vitro. Break formation was linear with dose up to 10 Krad,resulting in 0.27 double-strand breaks per kilorad per genome equivalentof DNA and requiring 1080 eV/double-strand break. The influence ofpossible non-linear DNA conformations of these calculations is discussed.Repair of ionizing radiation damage to folded chromosomes wasobserved within 2-3 hours of post-irradiation incubation in growth medium.A model based on recombinational repair is proposed to explain the formationof 2200-2300S material during early stages of incubation and subsequentchanges in the gradient profiles. Such behavior is not observedfor post-irradiation incubation of wild-type cells in buffer or for arecA-13 strain incubated in growth medium. Association of unrepaired DNAwith plasma membrane is proposed to explain the formation of a peak ofrapidly sedimenting material (>>3100S) during the later stages of repair.Direct evidence of repair of double-strand breaks during post-irradiationincubation in growth medium was obtained from gradientprofiles of DNA from RNAse-digested chromosomes. The sedimentationcoefficient of broken molecules was restored to the value of unirradiatedDNA after 2-3 hours of incubation, and the fraction of the DNA repaired inthis fashion was equal to the fraction of cells which survived at thesame dose. An average of 2.7 double-strand breaks per genome per lethalevent was observed, suggesting that 1-2 double-strand breaks per genomeare 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