Recombination Between Existing Homologous Chromosomes

Enchanced survival of ultraviolet-irradiated Escherichia coli dependent upon the recA + gene appears to result from the ability to seal gaps in newly synthesized deoxyribonucleic acid (DNA) produced when DNA containing pyrimidine dimers is replicated (8). Strains which are recombination-deficient (recA) do not seal these gaps and degrade their DNA to a considerable extent. Rupp and Howard-Flanders (8) postulated that the gap-sealing process involves genetic recombination between the two daughter chromosomes produced at replication. Recombination-deficient bacteria are also sensitive to ionizing radiation, although here the repair process dependent upon the recA + gene appears to involve the sealing of single-strand DNA breaks formed directly by the radiation (5) We may therefore ask whether recA+-dependent repair of ionizing radiation damage requires recombination between existing homologous chromosomes, i.e., before replication, analogous to that postulated to occur after replication of ultraviolet photoproducts. If such a hypothesis is correct, recA+ bacteria with but a single unreplicated chromosome should be as sensitive as recA bacteria, as there would be no possibility of recombination between homologous chromosomal regions. In testing this hypothesis, we found that, contrary to a widely held belief, stationary-phase bacteria rarely contained a single unreplicated chromosome. We grew E. coil B/r WP2 trp recA+ and a recA derivative in a chemostat at 37 C at a generation time of about 4.5 hr. The apparatus was that described by Baker (1) but with a covering of black paper to exclude light. The medium was M9 salts plus 100 ,ug of glucose/ml and 10 ;tg of tryptophan/ml. Plate count viability exceeded 90% for the recA+ strain. After cultures were run for at least 48 hr, bacteria were removed and suspended in phosphate buffer (0.067 M, pH 6.8) for irradiation with cobalt-60 gamma rays. According to Kubitschek, Bendigkeit, and Loken (6), bacteria grown in this way possess a single chromosome which replicates immediately before division. Measurements of DNA by the method of Burton (3) based on seven cultures indicated an average DNA content of 5.66 i 0.26 x 1015 g per bacterium. This corresponds to 1.23 times the amount of DNA in an unreplicated E. coli genome, assuming that one genome-equivalent is 2.8 x 109 daltons (4), or 4.6 x 1015 g of DNA. Therefore, at least 77% of the gehes in the population are present as only one copy per cell. If DNA replication occurs just before cell division, in an asynchronous population, approximately 50 to 70% (depending on the proportion of bacteria which have completed replication) of the bacteria would have no regions duplicated as a result of replication. Measurements of bacteria growing freely at a generation time of about 45 min in the same medium with nonlimiting glucose (0.4%) contained 1.11 ± 0.01 x 10-I' g of DNA per bacterium (based on four cultures). This is equivalent to about 2.4 genome-equivalents of DNA. If each nucleus contains 1/1 n2 (1.44) genome-