DNA Replication, Repair, and Mutagenesis in Permeable E. coli Cells That are Fully Viable
When suspended in an isotonic buffer E. coli cells repair radiation-induced DNA single-strand breaks by a very rapid, polA-dependent process (Town et al., 1971; Boye et al., 1974). Figure 1 shows the result of an experiment where intracellular, circular phage λ DNA molecules have been irradiated in wild type host cells suspended in different buffers. The cells were exposed to 8 krad of 4 MeV electrons and the fraction of broken circles was measured as a function of postirradiation incubation time at 37°C. As expected, repair was very rapid in the isotonic phosphate-buffered saline (PBS). However, rejoining is completely abolished in the hypotonic 40 mM Tris buffer. This loss of repair capacity depends on the tonicity of the buffer, since normal repair occurs in 40 mM Tris buffer containing 0.13 M NaC1 or 0.2 M sucrose (data not shown). When the Tris buffer is supplemented with all four deoxyribonucleoside triphosphates (dNTP’s), Mg2+, and NAD, the rapid repair seems to be restored (Fig. 1). This suggests that factors necessary for repair leak out of the cells during treatment with hypotonic buffer. Also, the cells remain permeable so that the necessary factors, when supplied to the suspending buffer, can penetrate into the cells.
KeywordsTris Buffer Hypotonic Buffer Sulfhydryl Compound Isotonic Buffer Norwegian Radium Hospital
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