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.