Molecular Insights into DNA Polymerase Deterrents for Ribonucleotide Insertion

Abstract

DNA polymerases can misinsert ribonucleotides that lead to genomic instability. DNA polymerase ␤ 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 (ϳ2.5 and 2.6 Å , respectively). Structure-based modeling indicates that the local energetic cost of positioning these closely spaced oxygens is ϳ2.2 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 where DNA polymerase ␤ utilizes two strategies, steric and geometric, with a single protein residue to deter ribonucleotide insertion.

Structural Factors That Determine Selectivity of a High Fidelity DNA Polymerase for Deoxy-, Dideoxy-, and Ribonucleotides

“…Molecular modeling suggested that this residue could interfere with binding of the rNTP 2Ј-OH and that reduction of its steric bulk accounts for the lowering of specificity in these mutants (14,28). Structural studies of a high (23) and low fidelity (29) DNA polymerase are consistent with this "steric gate" hypothesis.…”

Insights into the Conformation of Aminofluorene-Deoxyguanine Adduct in a DNA Polymerase Active Site

“…As observed previously (48,49), weak rCTP and rGTP insertion opposite template dG with all divalent metal ions was observed. The weak insertion of ribonucleotides by pol ␤ with unmodified DNA has been attributed to steric and electrostatic deterrents with Tyr-271 (59). Although in the -CG*A-sequence the insertion of rNTPs was not observed, weak insertion of CTP opposite dG-FAF in -TG*A-could be clearly seen (Fig.…”

The Structural Basis of the Kinetic Mechanism of a Gap-Filling X-Family DNA Polymerase That Binds Mg2+-dNTP Before Binding to DNA

“…15 Kinetic and structural studies of Polβ revealed that Tyr271 at the nucleotide-binding pocket contributes to reject ribonucleotide insertion with steric and geometric effects. 39 The backbone carbonyl of Tyr271 is clashed with the 2′-OH of the ribonucleotides, and the side chain of Tyr271 adjusts its catalytic positioning by interaction with the primer terminus. The conservation of this tyrosine in ttPolX (Tyr258; Figs.…”