Proflavine formed a complex with transforming deoxyribonucleic acid (DNA) from Haemophilus influenzae, with optimal formation at a ratio of proflavine to DNA of 0.06. The rate of dissociation of the complex by dialysis increased in the order: native, denatured, renatured DNA. The transforming activity of the DNA was reduced by its interaction with proflavine. This inactivation was dependent on the physical state of the DNA, the proflavine concentration, and the temperature. DNA that had been denatured and renatured was most sensitive; native DNA was much less sensitive. The inactivation remained after dialysis and was stable to prolonged storage. It is concluded that the inactivation of transforming DNA by proflavine takes place by a mechanism different from that of DNA-proflavine complex formation.

The lethal and mutagenic effects of H202 on wild-type Haemophilus influenzae Rd and on uvrl, uvr2, recl, and rec2 mutant strains were studied. The first two mutants are sensitive to UV, and the second two are defective in recombination. Rd, uvrl, and recl strains were more sensitive to the killing effect of H202 treatment than were uvr2 and rec2 strains. There were peaks of mutagenesis at two H202 concentrations over a range of 30 to 275 mM. Our results suggest a specific repair of H202 damage that is independent of the Uvr2 and Rec2 gene products. Sensitivity to the killing effect of H202 and to the lethal action of near-UV light were similar for Rd and uvrl strains. This finding suggests that the mechanisms of killing by and repair of H202 damage may have some overlap with those of near-UV radiation.We have been inter,..sted in the efi:ects of near-UV radiation (NUV) on Haemophilus influenzae and its transforming DNA (2, 3). Since NUV irradiation yields H202 that is lethal to Escherichia coli rec mutants (13, 17), we wished to examine whether the relationship between the biological effects of NUV and H202 (5) was the same for H. influenzae cells and transforming DNA.It has been observed that the in situ generation of H202 may be important in NUV killing of E. coli (8). Although some toxic effects of NUV may differ from those of H202, there is a synergistic effect between NUV and H202 (9). This information might be useful in understanding the mechanisms involved in damage and recovery (5). In E. coli, the katF or xthA mutation results in sensitivity to broad-spectrum NUV (14) as well as to H202 (5), independent of the recAl and uvrA6 mutations (15). Since mutations in the former genes do not render the cells sensitive to far UV (260 nm), this finding indicates that repair of NUV damage involves the enzyme exonuclease II, which differs from that of the SOS system.We now report the killing and mutagenic action of hydrogen peroxide in H. influenzae cells and compare the mechanisms of repair with those in E. coli.The bacterial strains used were wild-type H. influenzae Rd (6) and its uvrl, uvr2, recl, and rec2 mutants. The recl and rec2 mutations cause a defect in recombination, and they appear to be in different genes because the recl mutant is UV sensitive and the rec2 mutant is UV resistant (16). Mutations in the uvrl and uvr2 genes cause sensitivity to UV light. The products of both genes are required for excision of UV-induced pyrimidine dimers, but Uvr2 is believed to act later than Uvrl (11). The effect of H202 on these mutants can be related to the repair mechanisms of UV and H202 damage. Growth medium was brain heart infusion (Difco or Bioxon Laboratories) supplemented with the cofactors hemin and 3-nicotinamide adenine dinucleotide at 10 and 2 ,ug/ml, respectively (6). Hydrogen peroxide was purchased as a 30% aqueous solution from Merck. For each experiment, a fresh * Corresponding author. 1% solution and further dilutions of H202 were prepared in a solvent containing 0.10 M sodium chloride, 0.01 M phospha...