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.
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 > (6-4) > 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 > (6-4). 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.
Department of Chemistry, Washington University, St. Louis, Missouri 63130, USA.
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CA
Smith
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Baeten
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JS
Taylor
D001483:Base Sequence; D004247:DNA / Q000096:biosynthesis* / Q000528:radiation effects; D004261:DNA Replication / Q000528:radiation effects; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D015226:Dinucleoside Phosphates / Q000378:metabolism*; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000201:enzymology; D009841:Oligonucleotides / Q000378:metabolism / Q000528:radiation effects; D010777:Photochemistry; D012313:RNA / Q000096:biosynthesis* / Q000528:radiation effects; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D013329:Structure-Activity Relationship; D019914:Taq Polymerase / Q000378:metabolism; D014466:Ultraviolet Rays
D015226:Dinucleoside Phosphates; D009841:Oligonucleotides; C017758:thymidylyl-(3'-5')-thymidine; D012313:RNA; D004247:DNA; D019914:Taq Polymerase; C070331:Tli polymerase; D012194:RNA-Directed DNA Polymerase; D012321:DNA-Directed RNA Polymerases; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V