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Repair of oxidative DNA damage in gram-positive bacteria: the Lactococcus lactis Fpg protein
P Duwat1, R de Oliveira, S D Ehrlich
1Laboratoire de Génétique Microbienne, Institut National de la Recherche Agronomique, Jouy en Josas, France.
Abstract:
The formamidopyrimidine DNA glycosylase gene (fpg-L) of the Gram-positive microaerophilic bacterium Lactococcus lactis subsp. cremoris ML3 has been cloned, characterized and sequenced. The fpg-L gene is composed 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% identity with the Escherichia coli Fpg protein (Fpg-E). Polyclonal antibodies against Fpg-E react with the Fpg-L protein. The Fpg-L protein was purified to apparent homogeneity from the overproducing E. coli strain BH410 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 monomeric enzyme with a measured isoelectric point of 9.0. It contains one zinc per molecule and has a zinc finger motif localized at the carboxyterminal end (Cys-X2-Cys-X16-Cys-X2-Cys-X3-COOH). The Fpg-L protein has two enzyme activities: DNA glycosylase, which excises 2,6-diamino-4-hydroxy-5N-methylformamidopyrimidine and 7,8-dihydro-8-oxoguanine, and DNA nicking at abasic sites. Furthermore, the expression of the fpg-L gene in fpg and mutY mutants of E. coli suppresses their spontaneous GC-->TA mutator phenotype. The similarity of the activity of the two Fpg proteins and its conversation in evolutionarily distant bacteria may reflect the importance of its role in protecting bacterial DNA against oxidative free radicals.
Insights
Researchers cloned and characterized the formamidopyrimidine DNA glycosylase gene (fpg-L) from Lactococcus lactis. This enzyme repairs oxidative DNA damage, showing functional similarity to its E. coli counterpart.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- The formamidopyrimidine DNA glycosylase gene (fpg-L) in Lactococcus lactis subsp. cremoris ML3 was investigated.
- DNA glycosylases are crucial for DNA repair, particularly against oxidative damage.
Purpose of the Study:
- To clone, characterize, and sequence the fpg-L gene from Lactococcus lactis.
- To analyze the biochemical properties and DNA repair activities of the Fpg-L protein.
- To assess the functional conservation of Fpg-L in DNA repair pathways.
Main Methods:
- Gene cloning, sequencing, and protein expression in E. coli.
- Protein purification and characterization (molecular weight, isoelectric point, zinc content).
- Enzyme activity assays (DNA glycosylase and nicking activities).
- Complementation studies in E. coli mutants (fpg and mutY).
Main Results:
- The fpg-L gene (819 bp) encodes a 31.3 kDa protein (Fpg-L) with 59% similarity to E. coli Fpg.
- Purified Fpg-L is a 30 kDa monomeric enzyme with DNA glycosylase activity against specific oxidized bases and nicking activity at abasic sites.
- Fpg-L contains a zinc finger motif and suppresses the mutator phenotype in E. coli fpg and mutY mutants.
Conclusions:
- The Fpg-L protein from Lactococcus lactis possesses DNA repair functions similar to E. coli Fpg.
- The conserved activity suggests Fpg-L plays a vital role in protecting bacterial DNA from oxidative stress.
- This study highlights the evolutionary importance of DNA repair mechanisms across different bacterial species.