THE MUTATOR FORM OF POLYMERASE-BETA WITH AMINO-ACID SUBSTITUTION AT TYROSINE-265 IN THE HINGE REGION DISPLAYS AN INCREASE IN BOTH BASE SUBSTITUTION AND FRAME-SHIFT ERRORS

This study describes the first complete in vitro error specificity ana lysis of a mutator DNA polymerase that is altered in a residue not pre dicted to contact either the DNA or dNTP substrate. We examined this m utator form of polymerase beta (Y265C) in order to elucidate the criti cal role tyrosine 265 plays in the accuracy of DNA synthesis, Our resu lts demonstrate that an increase in both frame shift errors in homonuc leotide repeat sequences and base substitution errors contribute nearl y equally to the Y265C mutator phenotype. The models described for pro duction of these errors, primer/template misalignment and base misinco rporation, respectively, are distinctly different, suggesting the Y265 C alteration affects discrimination against both types of error produc tion pathways, In addition, Y265C displays a 530-fold increase in mult iple errors within the 203-base pair target region examined, relative to that of wild type. Processivity studies revealed that Y265C retains the near distributive nature of DNA synthesis characteristic of the w ild type polymerase beta. Therefore, multiple errors exhibited by Y265 C most likely result from independent polymerase binding events. Local ization of tyrosine 265 in the X-ray crystallographic structure sugges ts this residue may play a role in mediating a conformational change o f the polymerase [Pelletier, H., et al. (1996) Biochemistry 35, 12742- 12761]. A conformational change is predicted to enhance the accuracy o f DNA synthesis by imposing an induced fit selection against premutati onal intermediates. The observed loss of discrimination against both m isalignment-mediated and misincorporation-mediated errors produced by polymerase Y265C is consistent with such a model.