title: Roles of the active site residues and metal cofactors in noncanonical base-pairing during catalysis by human DNA polymerase iota.
abstract: Human DNA polymerase iota (Pol ι) is a Y-family polymerase that can bypass various DNA lesions but possesses very low fidelity of DNA synthesis in vitro. Structural analysis of Pol ι revealed a narrow active site that promotes noncanonical base-pairing during catalysis. To better understand the structure-function relationships in the active site of Pol ι we investigated substitutions of individual amino acid residues in its fingers domain that contact either the templating or the incoming nucleotide. Two of the substitutions, Y39A and Q59A, significantly decreased the catalytic activity but improved the fidelity of Pol ι. Surprisingly, in the presence of Mn(2+) ions, the wild-type and mutant Pol ι variants efficiently incorporated nucleotides opposite template purines containing modifications that disrupted either Hoogsteen or Watson-Crick base-pairing, suggesting that Pol ι may use various types of interactions during nucleotide addition. In contrast, in Mg(2+) reactions, wild-type Pol ι was dependent on Hoogsteen base-pairing, the Y39A mutant was essentially inactive, and the Q59A mutant promoted Watson-Crick interactions with template purines. The results suggest that Pol ι utilizes distinct mechanisms of nucleotide incorporation depending on the metal cofactor and reveal important roles of specific residues from the fingers domain in base-pairing and catalysis.
authors.affiliation: Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia. Electronic address: amakarova-img@yandex.ru.
authors.forename: Alena V
authors.initials: AV
authors.lastname: Makarova
authors.affiliation: Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia.
authors.forename: Artem
authors.initials: A
authors.lastname: Ignatov
authors.affiliation: Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia.
authors.forename: Nataliya
authors.initials: N
authors.lastname: Miropolskaya
authors.affiliation: Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia. Electronic address: akulb@img.ras.ru.
authors.forename: Andrey
authors.initials: A
authors.lastname: Kulbachinskiy
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D003067:Coenzymes / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D011485:Protein Binding; D011687:Purines / Q000378:metabolism; D000094769:DNA Polymerase iota
chemical_list: D003067:Coenzymes; D011687:Purines; D008345:Manganese; D004259:DNA-Directed DNA Polymerase; D008274:Magnesium; D000094769:DNA Polymerase iota; C000722070:POLI protein, human
keywords: DNA lesions; DNA polymerase iota; Hoogsteen base-pairing; Replication fidelity