title: Kinetic and Structural Impact of Metal Ions and Genetic Variations on Human DNA Polymerase ι.
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+-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,dCTPThe R96G and Δ1-25 variants displayed a 53-fold decrease and a 3-fold increase, respectively, in kpol/Kd,dCTPfor 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.
authors.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.
authors.forename: Jeong-Yun
authors.initials: JY
authors.lastname: Choi
authors.affiliation: the Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, and.
authors.forename: Amritaj
authors.initials: A
authors.lastname: Patra
authors.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.
authors.forename: Mina
authors.initials: M
authors.lastname: Yeom
authors.affiliation: the Department of New Biology, Daegu Gyeongbuk Institute of Science and Technology, Daegu 42988, Republic of Korea.
authors.forename: Young-Sam
authors.initials: YS
authors.lastname: Lee
authors.affiliation: the Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, and.
authors.forename: Qianqian
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: the Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, and.
authors.forename: Martin
authors.initials: M
authors.lastname: Egli
authors.affiliation: the Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, and f.guengerich@vanderbilt.edu.
authors.forename: F Peter
authors.initials: FP
authors.lastname: Guengerich
mesh_terms: D019943:Amino Acid Substitution; D018360:Crystallography, X-Ray; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry* / Q000378:metabolism; D008345:Manganese / Q000737:chemistry* / Q000378:metabolism; D020125:Mutation, Missense; D000072417:Protein Domains; D000094769:DNA Polymerase iota
chemical_list: D003845:Deoxycytosine Nucleotides; C024107:2'-deoxycytidine 5'-triphosphate; D008345:Manganese; D004259:DNA-Directed DNA Polymerase; D008274:Magnesium; D000094769:DNA Polymerase iota; C000722070:POLI protein, human
keywords: DNA enzyme; DNA polymerase; X-ray crystallography; crystal structure; enzyme kinetics; genetic variation; kinetics; metal ion-protein interaction; pre-steady-state kinetics; translesion DNA synthesis