3NDK

RB69 DNA Polymerase (Y567A) Ternary Complex with dCTP Opposite dG

Summary for 3NDK

• Entry DOI: 10.2210/pdb3ndk/pdb
• Related: 1IG9 3LZI 3LZJ 3NAE 3NCI 3NE6
• Descriptor: DNA polymerase, DNA (5'-D(*TP*CP*AP*GP*GP*TP*AP*AP*GP*CP*AP*GP*TP*CP*CP*GP*CP*G)-3'), DNA (5'-D(*GP*CP*GP*GP*AP*CP*TP*GP*CP*TP*TP*AP*(DOC))-3'), ... (6 entities in total)
• Functional Keywords: rb69 dna polymerase, fidelity, mutation frequencies, kinetics, transferase-dna complex, transferase/dna
• Biological source: Enterobacteria phage RB69 (Bacteriophage RB69)
• Total number of polymer chains: 3
• Total formula weight: 114803.93

Authors

Wang, M.,Wang, J.,Konigsberg, W.H. (deposition date: 2010-06-07, release date: 2011-01-26, Last modification date: 2023-09-06)

Primary citation

Xia, S.,Wang, M.,Lee, H.R.,Sinha, A.,Blaha, G.,Christian, T.,Wang, J.,Konigsberg, W.
Variation in Mutation Rates Caused by RB69pol Fidelity Mutants Can Be Rationalized on the Basis of Their Kinetic Behavior and Crystal Structures.
J.Mol.Biol., 406:558-570, 2011

PubMed Abstract: We have previously observed that stepwise replacement of amino acid residues in the nascent base-pair binding pocket of RB69 DNA polymerase (RB69pol) with Ala or Gly expanded the space in this pocket, resulting in a progressive increase in misincorporation. However, in vivo results with similar RB69pol nascent base-pair binding pocket mutants showed that mutation rates, as determined by the T4 phage rI forward assay and rII reversion assay, were significantly lower for the RB69pol S565G/Y567A double mutant than for the Y567A single mutant, the opposite of what we would have predicted. To investigate the reasons for this unexpected result, we have determined the pre-steady-state kinetic parameters and crystal structures of relevant ternary complexes. We found that the S565G/Y567A mutant generally had greater base selectivity than the Y567A mutant and that the kinetic parameters for dNMP insertion, excision of the 3'-terminal nucleotide residue, and primer extension beyond a mispair differed not only between these two mutants but also between the two highly mutable sequences in the T4 rI complementary strand. Comparison of the crystal structures of these two mutants with correct and incorrect incoming dNTPs provides insight into the unexpected increase in the fidelity of the S565G/Y567A double mutant. Taken together, the kinetic and structural results provide a basis for integrating and interpreting in vivo and in vitro observations.

PubMed: 21216248
DOI: 10.1016/j.jmb.2010.12.033

Experimental method

X-RAY DIFFRACTION (2 Å)