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.
Department of Biochemistry, University of Western Ontario, London, Ontario, Canada.
Kevin N
KN
Kirouac
Hong
H
Ling
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