Non‐cognate DNA damage prevents formation of active conformation of Y‐family DNA polymerases DinB and pol kappa

Abstract

Y‐family DNA polymerases are specialized to copy damaged DNA and are associated with an increased risk of mutagenesis due to their low fidelity. It is believed that the mechanism of nucleotide selection by Y‐family DNA polymerases involves conformational changes preceding nucleotidyl transfer but there is limited experimental evidence for such structural changes. Using hydrogen/deuterium exchange mass spectrometry, we demonstrate here that the Escherichia coli Y‐family DNA polymerase DinB and its human ortholog DNA polymerase κ undergo a conserved nucleotide‐induced conformational change in the presence of undamaged DNA and the correct incoming nucleotide. Notably, this holds true for damaged DNA containing N2‐furfuryl‐deoxyguanosine that is efficiently copied by these two polymerases, but not for damaged DNA containing the major groove modification O6‐methyl‐deoxyguanosine that is a poor substrate. Structural changes were localized to regions both near and far from the active site and involved several domains of the enzymes. Our observations suggest that DinB and pol κ utilize a common mechanism for nucleotide selection involving a conserved prechemical conformational transition promoted by the correct nucleotide and only preferred DNA substrates.