Minor groove interactions at the DNA polymerase beta active site modulate single-base deletion error rates

The structures of open and closed conformations of DNA polymerase beta (pol beta) suggests that the rate of single-nucleotide deletions during synthes is may be modulated by interactions in the DNA minor groove that align the templating base with the incoming dNTP. To test this hypothesis, we measure d the single-base deletion error rates of wild-type pol beta and lysine and alanine mutants of Arg(283), whose side chain interacts with the minor gro ove edge of the templating nucleotide at the active site. The error rates o f both mutant enzymes are increased > 100-fold relative to wild-type pol be ta, Template engineering experiments performed to distinguish among three p ossible models for deletion formation suggest that most deletions in repeti tive sequences by pol beta initiate by strand slippage. However, pol beta a lso generates deletions by a different mechanism that is strongly enhanced by the substitutions at Arg283. Analysis of error specificity suggests that this mechanism involves nucleotide misinsertion followed by primer relocat ion, creating a misaligned intermediate. The structure of pol beta bound to non-gapped DNA also indicates that the templating nucleotide and its downs tream neighbor are out of register in the open conformation and this could facilitate misalignment (dNTP or primer terminus) with the next template ba se.