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Incision at hypoxanthine residues in DNA by a mammalian homologue of the Escherichia coli antimutator enzyme endonuclease V.

ABSTRACT: Deamination of DNA bases can occur spontaneously, generating highly mutagenic lesions such as uracil and hypoxanthine. In Escherichia coli two enzymes initiate repair at hypoxanthine residues in DNA. The alkylbase DNA glycosylase, AlkA, initiates repair by removal of the damaged base, whereas endonuclease V, Endo V, hydrolyses the second phosphodiester bond 3' to the lesion. We have identified and characterised a mouse cDNA with striking homology to the E.coli nfi gene, which also has significant similarities to motifs required for catalytic activity of the UvrC endonuclease. The 37-kDa mouse enzyme (mEndo V) incises the DNA strand at the second phosphodiester bond 3' to hypoxanthine- and uracil-containing nucleotides. The activity of mEndo V is elevated on single-stranded DNA substrate in vitro. Expression of the mouse protein in a DNA repair-deficient E.coli alkA nfi strain suppresses its spontaneous mutator phenotype. We suggest that mEndo V initiates an alternative excision repair pathway for hypoxanthine removal. It thus appears that mEndo V has properties overlapping the function of alkylbase DNA glycosylase (Aag) in repair of deaminated adenine, which to some extent could explain the absence of phenotypic abnormalities associated with Aag knockout in mice.

SUBMITTER: Moe A

PROVIDER: S-EPMC167633 | biostudies-literature | 2003 Jul

REPOSITORIES: biostudies-literature

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Incision at hypoxanthine residues in DNA by a mammalian homologue of the Escherichia coli antimutator enzyme endonuclease V.

Moe Ane A Ringvoll Jeanette J Nordstrand Line M LM Eide Lars L Bjørås Magnar M Seeberg Erling E Rognes Torbjørn T Klungland Arne A

Nucleic acids research 20030701 14

Deamination of DNA bases can occur spontaneously, generating highly mutagenic lesions such as uracil and hypoxanthine. In Escherichia coli two enzymes initiate repair at hypoxanthine residues in DNA. The alkylbase DNA glycosylase, AlkA, initiates repair by removal of the damaged base, whereas endonuclease V, Endo V, hydrolyses the second phosphodiester bond 3' to the lesion. We have identified and characterised a mouse cDNA with striking homology to the E.coli nfi gene, which also has significan ...[more]

PMID: 12853604

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Project description:BackgroundHypoxanthine, prevalent in animals and plants, is used in the production of food additives, nucleoside antiviral drugs, and disease diagnosis. Current biological fermentation methods synthesize quantities insufficient to meet industrial demands. Therefore, this study aimed to develop a strain capable of industrial-scale production of hypoxanthine.ResultsDe novo synthesis of hypoxanthine was achieved by blocking the hypoxanthine decomposition pathway, thus alleviating transcriptional repression and multiple feedback inhibition, and introducing a purine operon from Bacillus subtilis to construct a chassis strain. The effects of knocking out the IMP(Inosine 5'-monophosphate) branch on the growth status and titer of the strain were then investigated, and the effectiveness of adenosine deaminase and adenine deaminase was verified. Overexpressing these enzymes created a dual pathway for hypoxanthine synthesis, enhancing the metabolic flow of hypoxanthine synthesis and preventing auxotrophic strain formation. Introducing IMP-specific 5' -nucleotidase addressed the issue of adenylate accumulation. In addition, the metabolic flow of the guanine branch was dynamically regulated by the guaB gene. The supply of glutamine and aspartic acid precursors was enhanced by introducing an exogenous glnA mutant gene, overexpressing aspC, and replacing the weaker promoter to regulate the aspartic acid branching pathway. Ultimately, fermentation in a 5 L bioreactor for 48 h produced 30.6 g/L hypoxanthine, with a maximum real-time productivity of 1.4 g/L/h, the highest value of hypoxanthine production by microbial fermentation reported so far.ConclusionsThe intracellular purine biosynthesis pathway is extensive and regulated at multiple levels in cells. The IMP branch in the hypoxanthine synthesis pathway has a higher metabolic flux. The current challenge lies in systematically allocating the metabolic flux within the branch pathway to achieve substantial product accumulation. In this study, E. coli was used as the chassis strain to construct a dual pathway for IMP and AMP(Adenosine 5'-monophosphate) synergistic hypoxanthine synthesis and dynamically regulate the guanine branch pathway. Overall, our experimental efforts culminated in a high-yield, plasmid- and defect-free engineered hypoxanthine strain.

Dataset Information

Incision at hypoxanthine residues in DNA by a mammalian homologue of the Escherichia coli antimutator enzyme endonuclease V.

Publications

Incision at hypoxanthine residues in DNA by a mammalian homologue of the Escherichia coli antimutator enzyme endonuclease V.

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