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Translesion DNA synthesis across non-DNA segments in cultured human cells

DNA lesions that have escaped DNA repair are tolerated via translesion DNA synthesis (TLS), carried out by specialized error-prone DNA polymerases. To evaluate the robustness of the TLS system in human cells, we examined its ability to cope with foreign non-DNA stretches of 3 or 12 methylene residues, using a gap-lesion plasmid assay system. We found that both the trimethylene and dodecamethylene inserts were bypassed with significant efficiencies in human cells, using both misinsertion and misalignment mechanisms. TLS across these non-DNA segments was aphidicolin-sensitive, and did not require polη. In vitro primer extension assays showed that purified polη, polκ and polι were each capable of inserting each of the four nucleotides opposite the trimethylene chain, but only polη and polκ could fully bypass it. Polη and polι, but not polκ, could also insert each of the four nucleotides opposite the dodecamethylene chain, but all three polymerases were severely blocked by this lesion. The ability of TLS polymerases to insert nucleotides opposite a hydrocarbon chain, despite the lack of any similarity to DNA, suggests that they may act via a mode of transient and local template-independent polymerase activity, and highlights the robustness of the TLS system in human cells. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png DNA Repair Elsevier
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