title: Noncognate DNA damage prevents the formation of the active conformation of the Y-family DNA polymerases DinB and DNA polymerase κ.
abstract: Y-family DNA polymerases are specialized to copy damaged DNA, and are associated with increased mutagenesis, owing 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. In particular, nucleotide-induced conformational changes in bacterial or eukaryotic Y-family DNA polymerases have, to date, not been extensively characterized. 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 N(2) -furfuryl-deoxyguanosine, which is efficiently copied by these two polymerases, but not for damaged DNA containing the major groove modification O(6) -methyl-deoxyguanosine, which is a poor substrate. Our observations suggest that DinB and DNA polymerase κ utilize a common mechanism for nucleotide selection involving a conserved prechemical conformational transition promoted by the correct nucleotide and only preferred DNA substrates.
authors.affiliation: Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA, USA.
authors.forename: Philip
authors.initials: P
authors.lastname: Nevin
authors.affiliation: Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA, USA.
authors.forename: Xueguang
authors.initials: X
authors.lastname: Lu
authors.affiliation: Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA, USA.
authors.forename: Ke
authors.initials: K
authors.lastname: Zhang
authors.affiliation: Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA, USA.
authors.forename: John R
authors.initials: JR
authors.lastname: Engen
authors.affiliation: Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA, USA.
authors.forename: Penny J
authors.initials: PJ
authors.lastname: Beuning
mesh_terms: D004249:DNA Damage / Q000502:physiology*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D041961:Deuterium Exchange Measurement; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D011487:Protein Conformation; D000099270:Y-Family DNA Polymerases
chemical_list: D004259:DNA-Directed DNA Polymerase; D029968:Escherichia coli Proteins; D006147:Guanine; C094399:DinB protein, E coli; C008449:O-(6)-methylguanine; C404302:POLK protein, human; D000099270:Y-Family DNA Polymerases
keywords: DNA replication; conformational change; hydrogen exchange mass spectrometry; nucleotide selection; substrate specificity