Fidelity of uracil-initiated base excision DNA repair in Escherichia coli cell extracts

The error frequency and mutational specificity associated with Escherichia coli uracil-initiated base excision repair were measured using an M13mp2 la cZ alpha DNA-based reversion assay. Repair was detected in cell-free extrac ts utilizing a form I DNA substrate containing a site-specific uracil resid ue. The rate and extent of complete uracil-DNA repair were measured using u racil-DNA glycosylase (Ung)- or double-strand uracil-DNA glycosylase (Dug)- proficient and -deficient isogenic E. coli cells. In reactions utilizing E. coli NR8051 (ung(+) dug(+)), similar to 80% of the uracil-DNA was repaired , whereas about 20% repair was observed using NR8052 (ung(-) dug(+)) cells, The Ung-deficient reaction was insensitive to inhibition by the PBS2 uraci l-DNA glycosylase inhibitor protein, implying the involvement of Dug activi ty. Under both conditions, repaired form I DNA accumulated in conjunction w ith limited DNA synthesis associated with a repair patch size of 1-20 nucle otides. Reactions conducted with E. coli BH156 (ung(-) dug(+)), BH157 (ung( +) dug(-)), and BH158 (ung(-) dug(-)) cells provided direct evidence for th e involvement of Dug in uracil-DNA repair. The rate of repair was B-fold gr eater in the Ung-proficient than in the Ung-deficient reactions, while repa ir was not detected in reactions deficient in both Ung and Dug. The base su bstitution reversion frequency associated with uracil-DNA repair was determ ined to be similar to5.5 x 10(-4) with transversion mutations dominating th e mutational spectrum. In the presence of Dug, inactivation of Ung resulted in up to a 7.3-fold increase in mutation frequency without a dramatic chan ge in mutational specificity.