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.
