Research Grants Program Office

To counteract the deleterious effects of DNA damage, cells are equipped with specialized polymerases to bypass DNA lesions. Previous biochemical studies revealed that DinB family DNA polymerases, including Escherichia coli DNA polymerase IV and human DNA polymerase κ, efficiently incorporate the correct nucleotide opposite some N(2)-modified 2'-deoxyguanosine derivatives. Herein, we used shuttle vector technology and demonstrated that deficiency in Polk or Poli in mouse embryonic fibroblast (MEF) cells resulted in elevated frequencies of G→T and G→A mutations at N(2)-(1-carboxyethyl)-2'-deoxyguanosine (N(2)-CEdG) and N(2)-carboxymethyl-2'-deoxyguanosine (N(2)-CMdG) sites. Steady-state kinetic measurements revealed that human DNA polymerase ι preferentially inserts the correct nucleotide, dCMP, opposite N(2)-CEdG lesions. In contrast, no mutation was found after the N(2)-CEdG- and N(2)-CMdG-bearing plasmids were replicated in POLH-deficient human cells or Rev3-deficient MEF cells. Together, our results revealed that, in mammalian cells, both polymerases κ and ι are necessary for the error-free bypass of N(2)-CEdG and N(2)-CMdG. However, in the absence of polymerase κ or ι, other translesion synthesis polymerase(s) could incorporate nucleotide(s) opposite these lesions but would do so inaccurately.