Formamidopyrimidine-DNA glycosylase of Escherichia coli: cloning and sequencing of the fpg structural gene and overproduction of the protein.

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RESUMO

An Escherichia coli genomic library composed of large DNA fragments (10-15 kb) was constructed using the plasmid pBR322 as vector. From it 700 clones were individually screened for increased excision of the ring-opened form of N7-methylguanine (2-6-diamino-4-hydroxy-5N-methyl-formamidopyrimidine) or Fapy. One clone overproduced the Fapy-DNA glycosylase activity by a factor of 10-fold as compared with the wild-type strain. The Fapy-DNA glycosylase overproducer character was associated with a 15-kb recombinant plasmid (pFPG10). After subcloning a 1.4-kb fragment which contained the Fapy-DNA glycosylase gene (fpg+) was inserted in the plasmids pUC18 and pUC19 yielding pFPG50 and pFPG60 respectively. The cells harbouring pFPG60 displayed a 50- to 100-fold increase in glycosylase activity and overexpressed a 31-kd protein. From these cells the Fapy-DNA glycosylase was purified to apparent physical homogeneity as evidenced by a single protein band at 31 kd on SDS-polyacrylamide gels. The amino acid composition of the protein and the amino acid sequence deduced from the nucleotide sequence demonstrate that the cloned fragment contains the structural gene coding for the Fapy-DNA glycosylase. The nucleotide sequence of the fpg gene is composed of 809 base pairs and codes for a protein of 269 amino acids with a calculated mol. wt of 30.2 kd.

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