PURIFICATION AND PROPERTIES OF WILD-TYPE AND EXONUCLEASE-DEFICIENT DNA-POLYMERASE-II FROM ESCHERICHIA-COLI

Wild-type DNA polymerase II (pol II and an exonuclease-deficient pol I I mutant (D155A/E157A) have been overexpressed and purified in high yi eld from Escherichia coli. Wild-type pol II exhibits a high proofreadi ng 3'-exonuclease to polymerase ratio, similar in magnitude to that ob served for bacteriophage T4 DNA polymerase. While copying a 250-nucleo tide region of the lacZ alpha gene, the fidelity of wild-type pol II i s high, with error rates for single-base substitution and frameshift e rrors being less than or equal to 10(-6). In contrast, the pol II exon uclease-deficient mutant generated a variety of base substitution and single base frameshift errors, as well as deletions between both perfe ct and imperfect directly repeated sequences separated by a few to hun dreds of nucleotides. Error rates for the pol II exonuclease-deficient mutant were from greater than or equal to 13- to greater than or equa l to 240-fold higher than for wild-type pol II, depending on the type of error considered. These data suggest that from 90 to >99% of base s ubstitutions, frameshifts, and large deletions are efficiently proofre ad by the enzyme. The results of these experiments together with recen t in vivo studies suggest an important role for pol II in the fidelity of DNA synthesis in cells.