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An improved infectivity assay for DNA repair

Most assays for DNA repair either measure a single biochemical end-point (e.g. dimer excision, 'repair synthesis' of DNA) or the biological end-point under conditions where repair and DNA replication are concomitant (e.g. cell survival, phage reactivation). For the biochemical dissection of DNA repair mechanisms, however, it is frequently desirable to uncouple repair from replication. The repressed infection system (Hays & Boehmer, 1978), originally used to study u.v.-stimulated recombination, accomplishes this. Essentially, Escherichia coli lysogens are superinfected with 'H-labelled phage. Cell samples are taken at known times after infection and whole cell DNA extracts are made. The DNA is further purified by phenol extraction and dialysed against Tris buffer, pH 8.0 lO.05 MTris/HCI (pH 8.0)/0.001 M-disodium-EDTA]. It can then be assayed either to a biological end-point, by determining its infectivity or to a biochemical end-point, e.g. by determining its thymine-dimer content. In order to measure infectivity more efficiently a spheroplast strain which is totally repair-deficient but transfects undamaged DNA efficiently was constructed. E. coli strain KL176/1 (Hays & Boehmer, 1978), a spheroplasting strain which is recA-, recB-/1 was transformed to Rec+ with plasmid pDR 1453 (Sancar & Rupp, 1979) which carries a functional rec A gene. A galkmutant of the transformant was selected by propagating on medium containing deoxygalactose. This strain was then transduced to galkt uvrBwith bacteriophage PI. Plasmid curing, by growing the strain overnight at 37°C in nutrient broth containing 5pg of coumermycin/ml, yielded the recA-,recB-,uorB-/l strain. Spheroplasts of this strain transfected unirradiated phage DNA as efficiently as the uvrB+ parent, but transfected irradiated phage DNA 300x less efficiently. Repair was measured either by determining the infectivity of irradiated phage DNA relative to unirradiated phage DNA which had also passed through a similar infection (relative infectivity) or by measuring the removal of thymine dimers. The effect of temperature on repair was studied and a comparison of results obtained with the infectivity and thymine-dimer assays was made. E. coli 1 lysogen JH158 was infected with either unirradiated or irradiated (100 J m-*, 254 nm) alM-l96538-ind-red, phage (Hays et al., 1981) at a multiplicity of infection of 15. Cell samples were removed at known intervals up to 240 minutes after infection, and DNA was extracted. The relative infectivity of the DNA was determined and the remainder was ethanolprecipitated. Precipitated DNA was resuspended in Hepes buffer, pH 7.6 10.05 M-Hepes (pH 7.6)/0.001 M-disodium EDTA/O.OOS M-MgCl,]. Thymine-dimer content was estimated by determining the number of strand-breaks generated by treatment with an excess of the Sephadex G-75 fraction of the Micrococcus luteus thymine-dimer-specific endonuclease preparation (Haseltine et al., 1980). The number of alkalisusceptible breaks per duplex molecule generated in enzyme