title: 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 wild-type 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 where DNA polymerase β utilizes two strategies, steric and geometric, with a single protein residue to deter ribonucleotide insertion.
authors: Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709-2233, USA.
authors: Nisha A
authors: NA
authors: Cavanaugh
authors: William A
authors: WA
authors: Beard
authors: Vinod K
authors: VK
authors: Batra
authors: Lalith
authors: L
authors: Perera
authors: Lee G
authors: LG
authors: Pedersen
authors: Samuel H
authors: SH
authors: Wilson
mesh_terms: D019943:Amino Acid Substitution; D020134:Catalytic Domain; D003460:Crystallization; D018360:Crystallography, X-Ray; D019951:DNA Polymerase beta / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006860:Hydrogen Bonding; D017434:Protein Structure, Tertiary; D012265:Ribonucleotides / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D012265:Ribonucleotides; D019951:DNA Polymerase beta