Effects of Coumarin, Pyronin Y, 6,9-Dimethyl 2-Methylthiopurine and Caffeine on Excision Repair and Recombination Repair in Escherichia coli

Abstract

S U M M A R YPyronin Y (0.06 and 0.16 mM), coumarin (2 and 4 mM), 6,pdimethyl 2-methylthiopurine (2 mM) and caffeine (8 m), strongly inhibit Uvr repair (presumed excision repair) of U.V. lesions in Escherichia coli. With 0.16 m-pyronin Y or 4m~-coumarin, one thymine dimer was a lethal event in the ~1 2 strain ~~2 9 2 6 rec A-13 and one to three dimers in the B/r derivative WP-2 rec A-13 and the B exr A derivative BS-2. Neither these compounds nor 8 mwcaffeine had a significant effect on the survival of irradiated bacteria of the corresponding uvr strains WP-2 Hcr-, BS-12, and BS-8. 1 suggest that 8 mM-caffeine, 0-16 mM-pyronin Y, 4 mM-coumarin and 2 m-6,9-dimethyl 2-methylthiopurine selectively block excision repair without substantially affecting recombination repair.Caffeine at 12 mM only slightly depressed the viability of a u.v.-irradiated culture of WP-2 Hcr-or BS-12, but at 16 mM there was a much greater effect, particularly at low U.V. doses ( < 40 ergs/mm2). Since excision repair appeared to be effectively blocked by 8 m-caffeine, the depression of U.V. survival of the uvr mutants by I 6 mM-Caffeine was presumed to be due to interference with recombination repair (Witkin & Farquharson, I 969). I N T R O D U C T I O NThe ultraviolet (u.v.) photoproducts of principal biological significance in Escherichia coli are pyrimidine dimers in DNA which interfere with normal DNA replication, and may lead to cell death (Setlow, 1968).Escherichia coli can cope with these lesions in the dark in at least two ways. In one, passage of the replicating fork past pyrimidine dimers in a priming strand results in a series of gaps in the freshly replicated strand, presumably because the dimers are unable to direct the insertion of nucleotides into the growing polynucleotide. The existence of discontinuities in the latter is thought to initiate recombination between daughter DNA strands permitting the reconstruction of the strands (Rupp & Howard-Flanders, 1968). This is referred to as ' recombination repair '. Two genes with alleles giving an impaired recombination repair effectiveness are rec A-13 and exr A . The other dark repair system involves the excision of pyrimidine dimers from the DNA and is termed excision repair. UVP Mutants are defective in this function.Some years ago caffeine (5 to 10 ITIM) was shown to inhibit dark repair (Witkin, 1958), and more recently excision repair (Setlow & Carrier, 1968). In vitro it neither inhibits DNA polymerase I activity (at a concentration of up to TOO mM, Grigg, 1968) (Kelly, Atkinson, Huberman & Kornberg, 1969). A number of factors other than caffeine interfere with dimer excision. These include crystal violet, acriflavine (Feiner & Hill, 1963;Setlow, 1964) cyanide, and starvation (Setlow & Carrier, 1968).The exact effects of caffeine on recombination repair are less obvious. Caffeine < 10.7 mM had very little effect on the U.V. survival of the uvr A mutant WP-2 Hcr-, which implies that the independent recombination-repair pathway was unaffected (Sideropoulos & Shankel, 1968;Wi...

Caffeine inhibits excision of 7-bromomethylbenz (a) anthracene-DNA adducts from exponentially growing but not from stationary phase Chinese hamster cells

“…In each case however, conditions (19), than elicit repair synthesis. The above observations confirm that caffeine per se does not influence excision repair in any direct fashion, although it has been suggested that high concentrations of caffeine inhibit excision repair in bacteria (20). The evidence presented in this report indicates that the excision of 7-BMBA induced lesions in a replicating cell is indeed inhibited by caffeine.…”

Caffeine suppresses homologous recombination through interference with RAD51-mediated joint molecule formation

“…Inhibitory effect of caffeine on recombination and recombinational repair in bacteria (61–63), yeast [meiotic (64) and mitotic (65)] and Drosophila (66) have been documented. As for mammalian cells, several previous studies suggested an effect of caffeine on homology-dependent repair (HDR) based on the use of the HDR assays and HR mutants in the context of induced damage (ionizing radiation or endonuclease) (38,40,67).…”

Growth and death of hela cells in the presence of caffeine

“…Such putative imbalance might take any of a variety of forms: for example, it could involve the uncoupling of processes that lead to the initiation of DNA synthesis (unpublished observations). Caffeine has been reported to perturb a multitude of metabolic processes; these include replication and repair of DNA in prokaryotic and eukaryotic cells (e.g., Grigg, 1968Grigg, , 1972Lehmann, 1972;Lehmann and Kirk-Bell, 1974;Tatsumi and Strauss, 1979;Painter, 1980) as well as the action of specific enzymes concerned with DNA synthesis, such as DNA polymerase I (Solberg et al, 19781, thymidine kinase (Sandlie et al, 19801, and a number of purine-metabolizing enzymes (Koch and Lamont, 1956;Nolan and Kidder, 1979). In addition, it binds to DNA, presumably in single-strand regions (Ts'o and Lu, 1964).…”