Structural basis of error-prone replication and stalling at a thymine base by human DNA polymerase iota.
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.
Kirouac
Kevin N
KN
Department of Biochemistry, University of Western Ontario, London, Ontario, Canada.
Ling
Hong
H
D000595:Amino Acid Sequence; D020029:Base Pairing; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D016415:Sequence Alignment; D013941:Thymine / Q000378:metabolism*; D000094769:DNA Polymerase iota
D009711:Nucleotides; D004259:DNA-Directed DNA Polymerase; D013941:Thymine; D000094769:DNA Polymerase iota; C000722070:POLI protein, human