REPAIR OF OXIDATIVE DNA-DAMAGE IN GRAM-POSITIVE BACTERIA - THE LACTOCOCCUS-LACTIS FPG PROTEIN

The formamidopyrimidine DNA glycosylase gene (fpg-L) of the Gram-posit ive microaerophilic bacterium Lactococcus lactis subsp. cremoris ML3 h as been cloned, characterized and sequenced. The fpg-L gene is compose d of 819 bp encoding a protein of 31.3 kDa (Fpg-L). The deduced amino acid sequence of the Fpg-L protein shows 59% similarity and 38% identi ty with the Escherichia coli Fpg protein (Fpg-E). Polyclonal antibodie s against Fpg-E react with the Fpg-L protein, The Fpg-L protein was pu rified to apparent homogeneity from the overproducing E. coli strain B H410 hosting plasmid pVE1064, which carries fpg-L under the control of the E. coli lac promoter. In its active form, Fpg-L is a 30 kDa monom eric enzyme with a measured isoelectric point of 9.0. It contains one zinc per molecule and has a zinc finger motif localized at the carboxy terminal end (Cys-X(2)-Cys-X(16)-Cys-X(2)-Cy5-X(3)-COOH). The Fpg-L pr otein has two enzyme activities: DNA glycosylase, which excises 2,6-di amino-4-hydroxy-5N-methylformamidopyrimidine and 7,8-dihydro-8-oxoguan ine, and DNA nicking at abasic sites. Furthermore, the expression of t he fpg-L gene in fpg and mutY mutants of E. coli suppresses their spon taneous GC --> TA mutator phenotype. The similarity of the activity of the two Fpg proteins and its conservation in evolutionarily distant b acteria may reflect the importance of its role in protecting bacterial DNA against oxidative free radicals.