Strand cleavage at psoralen adducts and pyrimidine dimers in DNA caused by interaction between semi-purified uvr+ gene products from Escherichia coli.
Partially purified extracts of Escherichia coli containing either uvrA+ or a mixture of uvrB+ and uvrC+ gene products were tested for an endonuclease activity on DNA treated with 8-methoxypsoralen plus 360-nm light. Neither of these fractions was active alone. The combined fractions, however, caused extensive strand cleavage of the psoralen-treated DNA. The endonuclease activity was dependent upon addition of ATP and Mg2+ to the reaction mixtures, and hence appeared similar to the UV-endonuclease activity previously shown to be reconstituted from the same fractions. It is concluded that the uvr+ gene products in these fractions interact to cause breakage of both psoralen-treated and UV-irradiated DNA. An examination of the dose-dependence relationship of the break formation in psoralen-treated DNA revealed that the enzyme acts upon psoralen mono-adducts. By varying the experimental conditions to increase the ratio of interstrand cross-links to mono-adducts it was found that the enzyme also acts upon cross-links, but with lower efficiency than for mono-adducts. Further studies of break formation in UV-irradiated DNA showed that elimination of pyrimidine dimers by treatment with photoreactivating enzyme in the light resulted in a loss of endonuclease-sensitive sites. This shows directly that pyrimidine dimers are the lesions recognized by the complemented uvr+ gene products in UV-irradiated DNA. For comparison, another endonuclease acting at pyrimidine dimers in DNA, the Micrococcus luteus UV-endonuclease, was also tested with psoralen-treated DNA, but no activity was observed. This and other data indicate that the repair endonuclease encoded by the uvr+ genes in E. coli is basically different from the other dimer-specific endonucleases previously characterized.
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D003086:Bacteriocin Plasmids; D004260:DNA Repair; D004270:DNA, Circular / Q000378:metabolism*; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D008027:Light; D008730:Methoxsalen; D011740:Pyrimidine Dimers / Q000378:metabolism; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D004270:DNA, Circular; D011740:Pyrimidine Dimers; D004720:Endonucleases; D008730:Methoxsalen