3RH5

DNA Polymerase Beta Mutant (Y271) with a dideoxy-terminated primer with an incoming deoxynucleotide (dCTP)

Summary for 3RH5

• Entry DOI: 10.2210/pdb3rh5/pdb
• Related: 3RH4 3RH6
• Descriptor: DNA polymerase beta, 5'-D(*CP*CP*GP*AP*CP*GP*CP*CP*GP*CP*AP*TP*CP*AP*GP*C)-3', 5'-D(*GP*CP*TP*GP*AP*TP*GP*CP*GP*(DDG))-3', ... (9 entities in total)
• Functional Keywords: dna polymerase beta mutant, nucleotide transferase, ribonucleotide insertion, dctp, transferase-dna complex, transferase/dna
• Biological source: Homo sapiens (human)
• Cellular location: Nucleus: P06746
• Total number of polymer chains: 4
• Total formula weight: 49141.90

Authors

Cavanaugh, N.A.,Beard, W.A.,Batra, V.K.,Perera, L.,Pedersen, L.G.,Wilson, S.H. (deposition date: 2011-04-11, release date: 2011-07-06, Last modification date: 2023-09-13)

Primary citation

Cavanaugh, N.A.,Beard, W.A.,Batra, V.K.,Perera, L.,Pedersen, L.G.,Wilson, S.H.
Molecular insights into DNA polymerase deterrents for ribonucleotide insertion.
J.Biol.Chem., 286:31650-31660, 2011

PubMed 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.

PubMed: 21733843
DOI: 10.1074/jbc.M111.253401

Experimental method

X-RAY DIFFRACTION (2.096 Å)