We measured the insertion fidelity of DNA polymerases alpha and beta and yeast DNA polymerase I at a template site that was previously observed to yield a high frequency of T----G transversions when copied by DNA polymerase beta but not by the other two polymerases. The results provide direct biochemical evidence that base substitution errors by DNA polymerase beta can result from a dislocation mechanism governed by DNA template-primer misalignment. In contrast to DNA polymerase beta, neither Drosophila DNA polymerase alpha nor yeast DNA polymerase I appear to misinsert nucleotides by a dislocation mechanism in either the genetic or kinetic fidelity assays. Dislocation errors by DNA polymerase beta are characterized primarily by a substantial reduction in the apparent Km for inserting a "correct," but ultimately errant, nucleotide compared to the apparent Km governing direct misinsertion. For synthesis by DNA polymerase beta, dislocation results in a 35-fold increase in dCMP incorporation opposite template T (T----G transversion) and a 20-35-fold increase in dTMP incorporation opposite T (T----A transversion); these results are consistent with parallel genetic fidelity measurements. DNA polymerase beta also produces base substitution errors by direct misinsertion. Here nucleotide insertion fidelity results from substantial differences in both Km and Vmax for correct versus incorrect substrates and is influenced strongly by local base sequence.

• PMID: 2661558
• PubMed
• PubTator

Reference Type

Comparative Study | Journal Article | Research Support, U.S. Gov't, P.H.S.

Authors

Boosalis MS, Mosbaugh DW, Hamatake R, Sugino A, Kunkel TA, Goodman MF

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