CIS-SYN THYMINE DIMERS ARE NOT ABSOLUTE BLOCKS TO REPLICATION BY DNA-POLYMERASE-I OF ESCHERICHIA-COLI INVITRO

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 ~0.15 unit/μL and 1-10 μM in each dNTP, termination one prior to the 3′-T of the dimer predominated. At 100 μM in each dNTP termination opposite the 3′-T of the dimer predominated and bypass occurred. Bypass at 100 μM in each dNTP depended on polymerase concentration, reaching a maximum of 20% in 1 h at approximately 0.2 unit/μL, 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 μM dATP:dNTP bias, 1% under dTTP bias, and an undetectable amount under either dGTP or dCTP bias. At 100 μM 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 >95% pdA was incorporated opposite both Ts of the dimer and that little or no frame shifting took place. A mechanism whereby products terminating in pdA opposite the 3′-T of the dimer are preferentially elongated by pol I was proposed to account for the higher sequence specificity of the bypass product than the termination product. On the basis of the results of this study, a mechanism was proposed that could account for the origin of the major mutation induced by ultraviolet light in bacteria, the C → T transition mutation at TpdC sites. © 1990, American Chemical Society. All rights reserved.