Molecular insights into DNA polymerase deterrents for ribonucleotide insertion

Abstract

DNA polymerases can misinsert ribonucleotides that lead to genomic instability. DNA polymerase β discourages ribonucleotide insertion with the backbone carbonyl of Tyr-271; alanine substitution of Tyr-271, but not Phe-272, resulted in a > 10-fold loss in discrimination. The Y271A mutant also inserted ribonucleotides more efficiently than wild type on a variety of ribonucleoside (rNMP)-containing DNA substrates. Substituting Mn 2+ for Mg 2+ decreased sugar discrimination for both wildtype and mutant enzymes primarily by increasing the affinity for rCTP. This facilitated crystallization of ternary substrate complexes of both the wild-type and Y271A mutant enzymes. Crystallographic structures of Y271A- and wild type-substrate complexes indicated that rCTP is well accommodated in the active site but that O2′ of rCTP and the carbonyl oxygen of Tyr-271 or Ala-271 are unusually close (∼2.5 and 2.6 Å , respectively). Structure-based modeling indicates that the local energetic cost of positioning these closely spaced oxygens is ∼2.2 kcal/mol for the wild-type enzyme. Because the side chain of Tyr-271 also hydrogen bonds with the primer terminus, loss of this interaction affects its catalytic positioning. Our results support a model whereDNApolymerase β utilizes two strategies, steric and geometric, with a single protein residue to deter ribonucleotide insertion.