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

Colin A. Smith, Mu Wang, Nan Jiang, Linda Che, Xiaodong Zhao, John Stephen Taylor

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79 Scopus citations

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 (<9%), the Dewar product more so (<33%), and the (6-4) product the most mutagenic (<73%). The mutation spectra were similar to those previously reported for the same photoproducts of the TT site of AGTTGG in the (+) strand of an M13 vector [Lawrence, C. W., et al. (1990) 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.

Original languageEnglish (US)
Pages (from-to)4146-4154
Number of pages9
JournalBiochemistry
Volume35
Issue number13
StatePublished - Apr 2 1996
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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