Repair of oxidative DNA damage in gram-positive bacteria: the Lactococcus lactis Fpg protein.
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.
Duwat
P
P
Laboratoire de Génétique Microbienne, Institut National de la Recherche Agronomique, Jouy en Josas, France.
de Oliveira
R
R
Ehrlich
S D
SD
Boiteux
S
S
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular; D003429:Cross Reactions; D004249:DNA Damage; D004260:DNA Repair / Q000235:genetics*; D043382:DNA-Formamidopyrimidine Glycosylase; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial / Q000235:genetics; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D013294:Lactococcus lactis / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics; D009699:N-Glycosyl Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D015032:Zinc / Q000032:analysis
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C024829:8-hydroxyguanine; D006147:Guanine; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli; D015032:Zinc