Abstract
The biological functions of human DNA polymerase (pol) θ, an A family polymerase, have remained poorly defined. Here we identify a role of polθ in translesion synthesis (TLS) in human cells. We show that TLS through the thymine glycol (TG) lesion, the most common oxidation product of thymine, occurs via two alternative pathways, in one of which, polymerases κ and ζ function together and mediate error-free TLS, whereas in the other, polθ functions in an error-prone manner. Human polθ is comprised of an N-terminal ATPase/helicase domain, a large central domain, and a C-terminal polymerase domain; however, we find that only the C-terminal polymerase domain is required for TLS opposite TG in human cells. In contrast to TLS mediated by polκ and polζ, in which polζ would elongate the chain from the TG:A base pair formed by polκ action, the ability of polθ alone to carry out the nucleotide insertion step, as well as the subsequent extension step that presents a considerable impediment due to displacement of the 5' template base, suggests that the polθ active site can accommodate highly distorting DNA lesions.
Highlights
Little is known of biological functions of DNA polymerase in human cells
In contrast to translesion synthesis (TLS) mediated by pol and pol, in which pol would elongate the chain from the thymine glycol (TG):A base pair formed by pol action, the ability of pol alone to carry out the nucleotide insertion step, as well as the subsequent extension step that presents a considerable impediment due to displacement of the 5 template base, suggests that the pol active site can accommodate highly distorting DNA lesions
Because the human cells used for these studies are proficient in nucleotide excision repair and base excision repair, a considerable number of TG lesions could have been removed by these DNA repair processes
Summary
Little is known of biological functions of DNA polymerase (pol) in human cells. Results: pol promotes replication through the common oxidation product, thymine glycol, in human cells. Opposite a cis-syn TT dimer, pol promotes error-free TLS, whereas polymerases and provide alternate pathways of mutagenic TLS [2] To verify whether these observations with the SV40 plasmid system reflect TLS mechanisms that operate during chromosomal replication, we determined the genetic control of error-free and. TG presents a strong block to synthesis by replicative and repair DNA polymerases; and we have shown previously that TLS opposite a TG lesion carried on the leading or lagging strand DNA template of SV40-based plasmid in human cells occurs in a predominantly error-free fashion and that polymerases and function together in promoting error-free replication through the lesion [1]. We provide evidence that TLS opposite the TG lesion in human cells is mediated by two alternate pathways, the polymerases /-dependent error-free pathway and another pathway in which pol alone functions in a mutagenic manner. We discuss the possible implications of this and other observations for TLS in human cells
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