Roger Hewitt

Reorientation of chromosome replication after exposure to ultraviolet light of Escherichia coli

Radioisotopic and density labeling methods have been used to examine the location of the site at which chromosome replication is resumed after the restoration of “net” DNA synthesizing activity in ultraviolet-irradiated cells. The findings were as follows. (1) Post-irradiation chromosome replication commences from a site other than the site that was active prior to exposure. (2) The extent of post-irradiation replication of the total DNA or of a chromosome region which was pulse-labeled immediately prior to exposure is limited to about 50%. (3) All chromosome regions do not have an equal probability of being duplicated following exposure, thus suggesting some specificity in the location of the site where replication is resumed. (4) The observation by others of a requirement for protein synthesis in the restoration of DNA synthesizing activity has been extended to show that inhibition of protein synthesis prevents the appearance of the new replication site. The findings have been interpreted in terms of the currently accepted model for the replication of the bacterial chromosome.

X-ray-induced perturbations in the replication of the bacterial chromosome.

Abstract Using [3H]thymine or [3H]thymidine as a pre-irradiation label and the thymine analogue 5-bromo-[14C]uracil as a combined density and radioisotopic label for observing post-irradiation DNA synthesis, it has been determined that relatively low doses of X-rays can induce changes in the normal sequence of chromosome replication. Changes are noted at doses of 2000 R or higher, with the effects becoming increasingly manifest with higher doses. The findings may be summarized as follows: (1) DNA synthesis immediately following irradiation occurs in limited regions of the chromosome. (2) Resumption of chromosome replication occurs at a point(s) apparently differing from the original growth point. (3) An early temporal appearance of a second “generation” of DNA is observed. (4) A portion of the DNA does not appear to participate in post-irradiation chromosomal replication. A feature of the experimental method was that at least 98 % of the cellular DNA was recovered in the CsCl gradients.

A fluorometric method for the detection of endodeoxyribonuclease on DNA-polyacrylamide gels

Abstract A fluorometric method has been described for the detection and identification of DNA-specific endonucleases in DNA-polyacrylamide gels after their separation by disk electrophoresis. This method was found sensitive enough to detect quantities as low as 5 pg of pancreatic DNase I. By the careful manipulation of the incubation conditions and the molecular state of the DNA substrate used, this method would be applicable to the detection and identification of other classes of enzymes exhibiting endo- and/or exonucleolytic activities such as those involved in the processes of replication, recombination, repair of DNA damage, and restriction.

Influence of ultraviolet irradiation on chromosome replication in ultraviolet-sensitive bacteria☆

Abstract We have studied the effect of ultraviolet irradiation upon chromosome replication in a strain of Escherichia coli that is unusually tolerant to density labeling with 5-bromouracil and that cannot excise thymine dimers ( E. coli C thy -321 uvrA 6). We find that the site and direction of replication are the same in irradiated and unirradiated cells during the period immediately following irradiation, and that all of the DNA synthesized during the first generation after exposure can be attributed to continued semi-conservative DNA replication. There was no evidence that ultraviolet light causes the premature initiation of a subsequent replication cycle. Indeed, initiation appeared to occur at nearly the same time in unirradiated and irradiated cells.

Concerning the dynamics of chromosome replication and degradation in a bacterial population exposed to X-rays

Abstract Successive radioisotopic labeling of the bacterial chromosome has been employed to determine whether the position of the label at the time of exposure to X-rays influences its pattern of replication subsequently. Following exposure to 5000 R of X-rays the chromosome region in front of the ‘replication-fork’ at the time of exposure is replicated to a greater extent than the region in or immediately behind the growth point. The latter region is prematurely replicated. If after X-ray exposure prevention of new cycles of DNA synthesis is accomplished by incubation of the cells in medium lacking required amino acids then the replication of all regions is markedly depressed. In addition an enhanced, non-selective degradation of chromosome regions is observed when irradiated cells are incubated without amino acids. Thus DNA in the vicinity of the replication-fork is degraded to the same extent as DNA located elsewhere. The data are interpreted as showing that following irradiation there exist two populations of chromosomes, the proportions of which are dose dependent. One population responds normally in regard to replication sequence and results from the resumption of semiconservative replication by the original replication-fork. The other, having lost its integrity, shows an aberrant type of replicative synthesis in which new sites of replication are initiated at random locations.

Unique forms of fast-sedimenting BU lambda DNA in a superinfected E. coli lysogen

Abstract When a lysogenic (immune) culture of Escherichia coli C thy -321 is superinfected with lambda phage in which 5-bromouracil (BU) has been substituted for the normal DNA base thymine, a large percentage of the linear BU lambda DNA injected into the cells is converted into a covalently closed circular monomer while a smaller amount is converted into DNA forms which sediment faster than the covalently closed circular monomer in both neutral and alkaline sucrose gradients. These unique BU-DNA structures form independently of coinfecting thymine lambda DNA, which forms only the covalently closed circular monomer. It has been found that the formation of the unique forms of fast-sedimenting BU lambda DNA is dependent upon the multiplicity of infection, i.e., the percent of unique forms is greatly reduced in cells superinfected at a multiplicity of infection of less than 1.0. This suggests that there is some type of atypical, intracellular interaction between two or more BU λ DNA molecules. Other observations concerning certain physical characteristics of these unique forms, such as their sedimentation properties in alkaline and neutral sucrose gradients following exposure to ultraviolet (uv) light, indicate that they are structures which differ from each other in molecular weight rather than in superhelical density.