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

Kinetic and Structural Impact of Metal Ions and Genetic Variations on Human DNA Polymerase iota
Choi, JY[Choi, Jeong-Yun]Patra, A[Patra, Amritaj]Yeom, M[Yeom, Mina]Lee, YS[Lee, Young-Sam]Zhang, QQ[Zhang, Qianqian]Egli, M[Egli, Martin]Guengerich, FP[Guengerich, F. Peter]
DGIST Authors
Lee, YS[Lee, Young-Sam]
Issue Date
Journal of Biological Chemistry, 291(40), 21063-+
Article Type
Amino AcidsCatalysisCatalyst ActivityCatalytic FunctionsCation-Pi InteractionsChainsCoordination ReactionsDistance GeometryGenesHuman Dna PolymeraseManganeseMetal IonsMetalsOctahedral Coordination GeometryPolymersStructural EffectStructural ImpactTranslesion Synthesis
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+-dependent and Mg2+-dependent activities of pol ι, whereas 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 with 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 analogue 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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
American Society for Biochemistry and Molecular Biology Inc.
Related Researcher
  • Author Lee, Young Sam Lab of genome maintenance
  • Research Interests DNA replication and repair; Restoration of cellular senescence; Structural and functional relationship of proteins
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