5KT3

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

(2016) J Biol Chem 291: 21063-21073

• PubMed: 27555320 Search on PubMedSearch on PubMed Central
• DOI: https://doi.org/10.1074/jbc.M116.748285
• Primary Citation of Related Structures:  
  5KT2, 5KT3, 5KT4, 5KT5, 5KT6, 5KT7


• PubMed Abstract: 
  

  DNA polymerase (pol) ι is a Y-family polymerase involved in translesion synthesis, exhibiting higher catalytic activity with Mn ²⁺ than Mg ²⁺ The human germline R96G variant impairs both Mn ²⁺ -dependent and Mg ²⁺ -dependent activities of pol ι, whereas the Δ1-25 variant selectively enhances its Mg ²⁺ -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 ²⁺ (0.15 mm) instead of Mg ²⁺ (2 mm) caused a 770-fold increase in efficiency (k _pol /K _{d ,dCTP} ) of pol ι for dCTP insertion opposite G, mainly due to a 450-fold decrease in K _{d ,dCTP} The R96G and Δ1-25 variants displayed a 53-fold decrease and a 3-fold increase, respectively, in k _pol /K _{d ,dCTP} for dCTP insertion opposite G with Mg ²⁺ when compared with wild type, substantially attenuated by substitution with Mn ²⁺ 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 ²⁺ or Mn ²⁺ , revealed that Mn ²⁺ achieves more optimal octahedral coordination geometry than Mg ²⁺ , 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.

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Organizational Affiliation: 

From the Division of Pharmacology, Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Gyeonggi-do 16419, Republic of Korea.