Structural basis of error-prone replication and stalling at a thymine base by human DNA polymerase iota

EMBO J. 2009 Jun 3;28(11):1644-54. doi: 10.1038/emboj.2009.122.

Abstract

Human DNA polymerase iota (pol iota) is a unique member of Y-family polymerases, which preferentially misincorporates nucleotides opposite thymines (T) and halts replication at T bases. The structural basis of the high error rates remains elusive. We present three crystal structures of pol complexed with DNA containing a thymine base, paired with correct or incorrect incoming nucleotides. A narrowed active site supports a pyrimidine to pyrimidine mismatch and excludes Watson-Crick base pairing by pol. The template thymine remains in an anti conformation irrespective of incoming nucleotides. Incoming ddATP adopts a syn conformation with reduced base stacking, whereas incorrect dGTP and dTTP maintain anti conformations with normal base stacking. Further stabilization of dGTP by H-bonding with Gln59 of the finger domain explains the preferential T to G mismatch. A template 'U-turn' is stabilized by pol and the methyl group of the thymine template, revealing the structural basis of T stalling. Our structural and domain-swapping experiments indicate that the finger domain is responsible for pol's high error rates on pyrimidines and determines the incorporation specificity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Pairing
  • Catalytic Domain
  • Crystallography, X-Ray
  • DNA Replication*
  • DNA-Directed DNA Polymerase / chemistry*
  • DNA-Directed DNA Polymerase / metabolism*
  • Humans
  • Models, Molecular
  • Molecular Sequence Data
  • Nucleotides / metabolism
  • Protein Binding
  • Protein Structure, Quaternary
  • Sequence Alignment
  • Thymine / metabolism*

Substances

  • Nucleotides
  • DNA polymerase iota
  • DNA-Directed DNA Polymerase
  • Thymine