Biophysical Journal 12: 1573-1582 (1972)
© 1972 the Biophysical Society

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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-³H (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 ³H-labeled strands in cells grown with BrdUrd at a much higher frequency than in ¹⁴C-labeled DNA of cells not exposed to BrdUrd. Breakage of the ³H-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 ³H-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-³H-labeled cells grown in the presence of BrdUrd eliminated 313 nm-induced breakage of the ³H-labeled strands. It is concluded that breakage of the ³H-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.