cis-syn thymine dimers are not absolute blocks to replication by DNA polymerase I of Escherichia coli in vitro.
Both Escherichia coli DNA polymerase I (pol I) and the large fragment of pol I (Klenow) were found to bypass a site-specific cis-syn thymine dimer, in vitro, under standard conditions. A template was constructed by ligating d(pCGTAT[c,s]TATGC), synthesized via a cis-syn thymine dimer phosphoramidite building block, to a 12-mer and 19-mer. The site and integrity of the dimer were verified by use of T4 denV endonuclease V. Extension of a 15-mer on the dimer-containing template by either pol I or Klenow led to dNTP and polymerase concentration dependent formation of termination and bypass products. At approximately 0.15 unit/microL and 1-10 microM in each dNTP, termination one prior to the 3'-T of the dimer predominated. At 100 microM in each dNTP termination opposite the 3'-T of the dimer predominated and bypass occurred. Bypass at 100 microM in each dNTP depended on polymerase concentration, reaching a maximum of 20% in 1 h at approximately 0.2 unit/microL, underscoring the importance of polymerase binding affinity for damaged primer-templates on bypass. Seven percent bypass in 1 h occurred under conditions of 100:10 microM dATP:dNTP bias, 1% under dTTP bias, and an undetectable amount under either dGTP or dCTP bias. At 100 microM in each dNTP, the ratio of pdA:pdG:pdC:pdT terminating opposite the 3'-T of the dimer was estimated to be 37:25:10:28. Sequencing of the bypass product produced under these conditions demonstrated that greater than 95% pdA was incorporated opposite both Ts of the dimer and that little or no frame shifting took place.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Chemistry, Washington University, St. Louis, Missouri 63130.
J S
JS
Taylor
C L
CL
O'Day
D001483:Base Sequence; D004256:DNA Polymerase I / Q000378:metabolism*; D004261:DNA Replication; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D011740:Pyrimidine Dimers / Q000378:metabolism*; D013698:Templates, Genetic
D009838:Oligodeoxyribonucleotides; D011740:Pyrimidine Dimers; D004256:DNA Polymerase I