We identified a dNTP substrate that is specific for only one DNA polymerase, pol κ. With this discovery we will able able to elucidate the functions of pol κ in a cell.
AS Prakasha Gowda, Marietta Lee, Thomas E. Spratt (2017) N2-Substituted 2′-Deoxyguanosine Triphosphate Derivatives, Selective Substrates for Human DNA Polymerase κ” Angew Chemie Intl Ed (2017) in press http://dx.doi.org/10.1002/anie.201611607.
Proposed mechanism by which N2-EBndG is incorporated into the DNA in cells
N2-Alkyl-2’-deoxyguanosine triphosphate (N2-alkyl-dGTP) derivatives with methyl, butyl, benzyl, 4-ethynylbenzyl substituents were prepared and tested for substrates for human DNA polymerases. N2-Benzyl-dGTP was equal to dGTP as a substrate for DNA polymerase κ, but was a poor substrate for pol b ,d, h, i ,or n. In vivo reactivity was evaluated by incubation of N2-4-ethynylbenzyl-dG with wild-type and pol κ deficient mouse embryonic fibroblasts. CuAAC click reaction with 5(6)-FAM-azide demonstrate that only pol κ containing cells were able to incorporate N2-4-ethynylbenzyl-dG into the nucleus. This is the first instance of a Y-family-polymerase-specific dNTP and this method can be used to probe the activity of pol κ in vivo.