title: Steric gate residues of Y-family DNA polymerases DinB and pol kappa are crucial for dNTP-induced conformational change.
abstract: Discrimination against ribonucleotides by DNA polymerases is critical to preserve DNA integrity. For many DNA polymerases, including those of the Y family, rNTP discrimination has been attributed to steric clashes between a residue near the active site, the steric gate, and the 2'-hydroxyl of the incoming rNTP. Here we used hydrogen/deuterium exchange (HDX) mass spectrometry (MS) to probe the effects of the steric gate in the Y-family DNA polymerases Escherichia coli DinB and human DNA pol κ. Formation of a ternary complex with a G:dCTP base pair in the active site resulted in slower hydrogen exchange relative to a ternary complex with G:rCTP in the active site. The protection from exchange was localized to regions both distal and proximal to the active site, suggesting that DinB and DNA pol κ adopt different conformations depending on the sugar of the incoming nucleotide. In contrast, when the respective steric gate residues were mutated to alanine, the differences in HDX between the dNTP- and rNTP-bound ternary complexes were attenuated such that for DinB(F13A) and pol κ(Y112A), ternary complexes with either G:dCTP or G:rCTP base pairs had similar HDX profiles. Furthermore, the HDX in these ternary complexes resembled that of the rCTP-bound state rather than the dCTP-bound state of the wild-type enzymes. Primer extension assays confirmed that DinB(F13A) and pol κ(Y112A) do not discriminate against rNTPs to the same extent as the wild-type enzymes. Our observations indicate that the steric gate is crucial for rNTP discrimination because of its role in specifically promoting a dNTP-induced conformational change and that rNTP discrimination occurs in a relatively closed state of the polymerases.
authors: Department of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, USA.
authors: Philip
authors: P
authors: Nevin
authors: Department of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, USA.
authors: John R
authors: JR
authors: Engen
authors: Department of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, USA. Electronic address: beuning@neu.edu.
authors: Penny J
authors: PJ
authors: Beuning
mesh_terms: D020134:Catalytic Domain; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009154:Mutation; D012265:Ribonucleotides / Q000378:metabolism; D013379:Substrate Specificity; D000099270:Y-Family DNA Polymerases
chemical_list: D004259:DNA-Directed DNA Polymerase; D003854:Deoxyribonucleotides; D029968:Escherichia coli Proteins; D012265:Ribonucleotides; C094399:DinB protein, E coli; C404302:POLK protein, human; D000099270:Y-Family DNA Polymerases
keywords: DNA replication; Enzyme specificity; Hydrogen exchange mass spectrometry; Nucleotide selection; Ribonucleotide