During base excision repair, DNA polymerase β fills 1–6-nucleotide gaps processively, reflecting a contribution of both its 8- and 31-kDa domains to DNA binding. Here we report the fidelity of pol β during synthesis to fill gaps of 1, 5, 6, or >300 nucleotides. Error rates during distributive synthesis by recombinant rat and human polymerase (pol) β with a 390-base gap are similar to each other and to previous values with pol β purified from tissues. The base substitution fidelity of human pol β when processively filling a 5-nucleotide gap is similar to that with a 361-nucleotide gap, but “closely-spaced” substitutions are produced at a rate at least 60-fold higher than for distributive synthesis. Base substitution fidelity when filling a 1-nucleotide gap is higher than when filling a 5-nucleotide gap, suggesting a contribution of the 8-kDa domain to the dNTP binding pocket and/or a difference in base stacking or DNA structure imposed by pol β. Nonetheless, 1-nucleotide gap filling is inaccurate, even generating complex substitution-addition errors. Finally, the single-base deletion error rate during processive synthesis to fill a 6-nucleotide gap is indistinguishable from that of distributive synthesis to fill a 390-nucleotide gap. Thus the mechanism of processivity by pol β does not allow the enzyme to suppress template misalignments.
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