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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

Differences in transformation of repair-deficient mutants of E. coli with BPDE- or chlorozotocin-modified plasmid DNA.

Plasmid pBR322 was alkylated with either chlorozotocin or with r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo-[a]pyrene (BPDE) before it was transformed into various strains of Escherichia coli. Plasmid survival was determined as ability to convert the bacteria to tetracycline and ampicillin resistance. Increased levels of alkylation caused a decrease in transforming activity in all strains studied. This decrease did not seem to be a result of alkylation induced strand scission, but rather some other biochemical or conformational change induced by the alkylating event. In E. coli AB1157 transformation was decreased by 50% with 6 alkylations/plasmid molecule for BPDE and 8-9 alkylations for chlorozotocin. At these levels of alkylation the loss in supercoiled DNA due to strand scission was less than 5%. Alkylated pBR322 was also transformed into repair-deficient strains of E. coli. In strain JC2924 (recA6) the survival of both BPDE- and chlorozotocin-modified DNA was similar to survival in the repair proficient strain AB1157, which would indicate that postreplicational repair of BPDE- or chlorozotocin-modified plasmid DNA was not significant under these conditions. Chlorozotocin-modified pBR322 did not seem to be repaired by the bacterial uvr-endonucleases as determined by plasmid survival in strains AB1884 (uvrC34), AB1885 (uvrB5) and AB1886 (uvrA6). With BPDE-alkylated plasmid DNA the results were strikingly different. Strains AB1884 and AB1886 were more sensitive to BPDE modified DNA than the wild type strain AB1157. Strain AB1885 was similar to AB1157 in sensitivity to BPDE-alkylated plasmid. These findings suggest that bacterial uvr-endonucleases may be able to recognize and repair BPDE-alkylated pBR322. The role of the uvrB protein in repair of alkylated DNA needs to be further investigated.[1]


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