Escherichia coli endonuclease VIII: cloning, sequencing, and overexpression of the nei structural gene and characterization of nei and nei nth mutants

DOI Web Site 被引用文献6件
  • D Jiang
    Department of Microbiology and Molecular Genetics, The University of Vermont, Burlington 05405, USA.
  • Z Hatahet
    Department of Microbiology and Molecular Genetics, The University of Vermont, Burlington 05405, USA.
  • J O Blaisdell
    Department of Microbiology and Molecular Genetics, The University of Vermont, Burlington 05405, USA.
  • R J Melamede
    Department of Microbiology and Molecular Genetics, The University of Vermont, Burlington 05405, USA.
  • S S Wallace
    Department of Microbiology and Molecular Genetics, The University of Vermont, Burlington 05405, USA.

書誌事項

公開日
1997-06
権利情報
  • https://journals.asm.org/non-commercial-tdm-license
DOI
  • 10.1128/jb.179.11.3773-3782.1997
公開者
American Society for Microbiology

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<jats:p>Escherichia coli possesses two DNA glycosylase/apurinic lyase activities with overlapping substrate specificities, endonuclease III and endonuclease VIII, that recognize and remove oxidized pyrimidines from DNA. Endonuclease III is encoded by the nth gene. Endonuclease VIII has now been purified to apparent homogeneity, and the gene, nei, has been cloned by using reverse genetics. The gene nei is located at 16 min on the E. coli chromosome and encodes a 263-amino-acid protein which shows significant homology in the N-terminal and C-terminal regions to five bacterial Fpg proteins. A nei partial deletion replacement mutant was constructed, and deletion of nei was confirmed by genomic PCR, activity analysis, and Western blot analysis. nth nei double mutants were hypersensitive to ionizing radiation and hydrogen peroxide but not as sensitive as mutants devoid of base excision repair (xth nfo). Single nth mutants exhibited wild-type sensitivity to X rays, while nei mutants were consistently slightly more sensitive than the wild type. Double mutants lacking both endonucleases III and VIII exhibited a strong spontaneous mutator phenotype (about 20-fold) as determined by a rifampin forward mutation assay. In contrast to nth mutants, which showed a weak mutator phenotype, nei single mutants behaved as the wild type.</jats:p>

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