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J Biol Chem, Vol. 273, Issue 34, 21933-21940, August 21, 1998

The Ability of a Variety of Polymerases to Synthesize Past Site-specific cis-syn, trans-syn-II, (6-4), and Dewar Photoproducts of Thymidylyl-(3'5')-thymidine

From the Department of Chemistry, Washington University, St. Louis, Missouri 63130

The role of photoproduct structure, 3'  5' exonuclease activity, and processivity on polynucleotide synthesis past photoproducts of thymidylyl-(3'  5')-thymidine was investigated. Both Moloney murine leukemia virus reverse transcriptase and 3'  5' exonuclease-deficient (exo) Vent polymerase were blocked by all photoproducts, whereas Taq polymerase could slowly bypass the cis-syn dimer. T7 RNA polymerase was able to bypass all the photoproducts in the order cis-syn > Dewar > () > trans-syn-II. Klenow fragment could not bypass any of the photoproducts, but an exo mutant could bypass the cis-syn dimer to a greater extent than the others. Likewise T7 DNA polymerase, composed of the T7 gene 5 protein and Escherichia coli thioredoxin, was blocked by all the photoproducts, but the exo mutant Sequenase 2.0 was able to bypass them all in the order cis-syn > Dewar > trans-syn-II > (). No bypass occurred with an exo gene 5 protein in the absence of the thioredoxin processivity factor. Bypass of the cis-syn and trans-syn-II products by Sequenase 2.0 was essentially non-mutagenic, whereas about 20% dTMP was inserted opposite the 5'-T of the Dewar photoproduct. A mechanism involving a transient abasic site is proposed to account for the preferential incorporation of dAMP opposite the 3'-T of the photoproducts.

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