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 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 ? utilizes two strategies, steric and geometric, with a single protein residue to deter ribonucleotide insertion.

SUBMITTER: Cavanaugh NA 

PROVIDER: S-EPMC3173102 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature