Mutation Spectra of M13 Vectors Containing Site-Specific Cis-Syn, Trans-Syn-I, (6−4), and Dewar Pyrimidone Photoproducts of Thymidylyl-(3‘→5‘)-Thymidine in Escherichia coli under SOS Conditions

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Abstract
The mutation spectra of cis-syn, trans-syn-I, (6−4), and Dewar pyrimidone photoproducts of the TT site of AATTAA and TATTAT in the (−) strand of a heteroduplex M13 vector were obtained in an excision and photoreversal repair deficient Escherichia coli host under SOS conditions. Oligonucleotides containing site-specific photoproducts were annealed to a complementary uracil-containing (+) strand that contained one or more unique pairs of nucleotide mismatches and used to prime (−) strand synthesis with a DNA polymerase and dNTPs. Following DNA synthesis, the reaction mixtures were incubated with T4 DNA ligase and ATP and then used to transfect SOS-induced competent CSRO6F‘ cells (uvrA6 and phr-1). The transfectants were plated, gridded, and probed by oligonucleotides specific for progeny of the (−) and (+) strands. Individual progeny of the photoproduct-containing (−) strands were plaque purified and sequenced by the dideoxy method. The cis-syn and trans-syn-I dimers were found not to be very mutagenic (Mol. Gen. Genet. 222, 166−168; LeClerc, J. E., et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 9685−9689] except that −1 deletion mutations were not observed for the trans-syn-I photoproducts, and a lower frequency of 3‘-T→C mutations was observed for the (6−4) photoproduct. Evidence that a small percentage of (+) strand repair of a double mismatch to the 3‘-side of the photoproduct site was obtained from transfection experiments in which a second double mismatch was introduced opposite or flanking the photoproduct. Analysis of the minor tandem mutations induced by the (6−4) and Dewar products suggests that the SOS polymerase complex is able to elongate what amounts to double mismatches opposite these photoproducts and is consistent with the action of a highly processive polymerase that lacks proofreading ability.