Breakage of parental DNA strands in Haemophilus influenzae by 313 nm radiation after replication in the presence of 5-bromodeoxyuridine

Abstract

Haemophilus influenzae was labeled with thymidine-(3)H (dThd), then grown in the presence of 5-bromodeoxyuridine (BrdUrd), and then irradiated with 313 nm light (a wavelength that selectively photolyzes DNA containing 5-bromouracil [BrUra]). Irradiation with 313 nm light induced breaks in the (3)H-labeled strands in cells grown with BrdUrd at a much higher frequency than in (14)C-labeled DNA of cells not exposed to BrdUrd. Breakage of the (3)H-labeled strands was about 0.6% as efficient as that of fully BrUra-substituted DNA. During growth in the presence of BrdUrd, susceptibility to 313 nm-induced breakage of the (3)H-labeled DNA strands increased, reaching a maximum in about one generation, and it decreased to zero during subsequent growth for one generation in medium containing dThd instead of BrdUrd. Heat denaturation of DNA extracted from dThd-(3)H-labeled cells grown in the presence of BrdUrd eliminated 313 nm-induced breakage of the (3)H-labeled strands. It is concluded that breakage of the (3)H-labeled DNA strands resulted from reaction with photoproducts in the base-paired, BrUra-containing strands, rather than from photolysis of BrdUrd incorporated into parental strands. It may be possible to utilize the phenomenon of interstrand breakage in physical studies of DNA replication.