ENDONUCLEASE-V (NFI) MUTANT OF ESCHERICHIA-COLI K-12

Endonuclease V (deoxyinosine 3' endonuclease), the product of the nfi gene, has a specificity that encompasses DNAs containing dIMP, abasic sites, base mismatches, uracil, and even untreated single-stranded DNA . To determine its importance in DNA repair pathways, nfi insertion mu tants and overproducers (strains bearing nfi plasmids) were constructe d, The mutants displayed a twofold increase in spontaneous mutations f or several markers and an increased sensitivity to killing by bleomyci n and nitrofurantoin, An nfi mutation increased both cellular resistan ce to and mutability by nitrous acid, This agent should generate poten tial cleavage sites for tile enzyme bg deaminating dAMP and dCMP in DN A to dIMP and dUMP, respectively, Relative to that of a wild-type stra in, an nfi mutant displayed a 12- to 1,000-fold increase in the freque ncy of nitrite-induced mutations to streptomycin resistance, which are known to occur in A.T base pairs, An nfi mutation also enhanced the l ethality caused by a combined deficiency of exonuclease III and dUTPas e, which has bern attributed to unrepaired abasic sites. However, neit her the deficiency nor the overproduction of endonuclease V affected t he growth of the single-stranded DNA phages M13 or phi X174 nor of Ura cil-containing bacteriophage lambda. These results suggest that endonu clease V has a significant role in the repair of deaminated deoxyadeno sine (deoxyinosine) and abasic sites in DNA, but there was no evidence for its cleavage in vivo of single-stranded or uracil-containing DNA.