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.
The mutations 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. Evidence that a small percentage of (+) strand repair of a double mismatch to the 3'-side 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.
Smith
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CA
Department of Chemistry, Washington University, St. Louis, Missouri 6330, USA.
Wang
M
M
Jiang
N
N
Che
L
L
Zhao
X
X
Taylor
J S
JS
D017104:Bacteriophage M13 / Q000235:genetics; D001483:Base Sequence; D004249:DNA Damage; D004269:DNA, Bacterial / Q000096:biosynthesis* / Q000737:chemistry / Q000528:radiation effects; D015226:Dinucleoside Phosphates / Q000528:radiation effects; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009692:Nucleic Acid Heteroduplexes / Q000096:biosynthesis / Q000737:chemistry; D009838:Oligodeoxyribonucleotides; D015345:Oligonucleotide Probes; D015183:Restriction Mapping; D013014:SOS Response, Genetics; D014162:Transfection; D014466:Ultraviolet Rays
D004269:DNA, Bacterial; D015226:Dinucleoside Phosphates; D009692:Nucleic Acid Heteroduplexes; D009838:Oligodeoxyribonucleotides; D015345:Oligonucleotide Probes; C017758:thymidylyl-(3'-5')-thymidine