SNAP output for PDB entry 5kt7 [SNAP web server]

PDB-id

5kt7; SNAP-derived features in text and JSON formats; DNAproDB

Class

transferase

Method

X-ray (3.151 Å)

Summary

Teranry complex of human DNA polymerase iota(1-445) inserting dcmpnpp opposite template g in the presence of mn2+

Reference

Choi JY, Patra A, Yeom M, Lee YS, Zhang Q, Egli M, Guengerich FP (2016): "Kinetic and Structural Impact of Metal Ions and Genetic Variations on Human DNA Polymerase iota." J.Biol.Chem., 291, 21063-21073. doi: 10.1074/jbc.M116.748285.

Abstract

DNA polymerase (pol) ι is a Y-family polymerase involved in translesion synthesis, exhibiting higher catalytic activity with Mn2+ than Mg2+. The human germline R96G variant impairs both Mn2+- and Mg2+-dependent activities of pol ι, while the Δ1-25 variant selectively enhances its Mg2+-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 Mn2+ (0.15 mM) instead of Mg2+ (2 mM) caused a 770-fold increase in efficiency (kpol/Kd,dCTP) of pol ι for dCTP insertion opposite G, mainly due to a 450-fold decrease in Kd,dCTP. The R96G and Δ1-25 variants displayed a 53-fold decrease and a 3-fold increase, respectively, in kpol/Kd,dCTP for dCTP insertion opposite G with Mg2+ when compared to wild-type, substantially attenuated by substitution with Mn2+. Crystal structures of pol ι ternary complexes, including the primer terminus 3'-OH and a non-hydrolyzable dCTP analog opposite G with the active-site Mg2+ or Mn2+, revealed that Mn2+ achieves more optimal octahedral coordination geometry than Mg2+, 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.