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Kinetic and Structural Impact of Metal Ions and Genetic Variations on Human DNA Polymerase ι.

Choi JY, Patra A, Yeom M, Lee YS, Zhang Q, Egli M, Guengerich FP
J Biol Chem. 2016 291 (40): 21063-21073

PMID: 27555320 · PMCID: PMC5076516 · DOI:10.1074/jbc.M116.748285

DNA polymerase (pol) ι is a Y-family polymerase involved in translesion synthesis, exhibiting higher catalytic activity with Mn than Mg The human germline R96G variant impairs both Mn-dependent and Mg-dependent activities of pol ι, whereas the Δ1-25 variant selectively enhances its Mg-dependent activity. We analyzed pre-steady-state kinetic and structural effects of these two metal ions and genetic variations on pol ι using pol ι core (residues 1-445) proteins. The presence of Mn (0.15 mm) instead of Mg (2 mm) caused a 770-fold increase in efficiency (k/K) of pol ι for dCTP insertion opposite G, mainly due to a 450-fold decrease in K The R96G and Δ1-25 variants displayed a 53-fold decrease and a 3-fold increase, respectively, in k/K for dCTP insertion opposite G with Mg when compared with wild type, substantially attenuated by substitution with Mn Crystal structures of pol ι ternary complexes, including the primer terminus 3'-OH and a non-hydrolyzable dCTP analogue opposite G with the active-site Mg or Mn, revealed that Mn achieves more optimal octahedral coordination geometry than Mg, with lower values in average coordination distance geometry in the catalytic metal A-site. Crystal structures of R96G revealed the loss of three H-bonds of residues Gly-96 and Tyr-93 with an incoming dNTP, due to the lack of an arginine, as well as a destabilized Tyr-93 side chain secondary to the loss of a cation-π interaction between both side chains. These results provide a mechanistic basis for alteration in pol ι catalytic function with coordinating metals and genetic variation.

© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

MeSH Terms (11)

Amino Acid Substitution Crystallography, X-Ray Deoxycytosine Nucleotides DNA-Directed DNA Polymerase Humans Hydrogen Bonding Kinetics Magnesium Manganese Mutation, Missense Protein Domains

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