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Helix-hairpin-helix protein MJ1434 from Methanocaldococcus jannaschii and EndoIV homologue TTC0482 from Thermus thermophilus HB27 do not process DNA uracil residues.

Schomacher L, Smolorz S, Ciirdaeva E, Ber S, Kramer W, Fritz HJ - Nucleic Acids Res. (2010)

Bottom Line: Sequence homologues of both proteins can be found throughout the archaeal domain of life.We propose that the uracil processing activities formerly found were due to contaminations with Ung enzyme.Use of Deltaung-strains as hosts for production of putatively DNA-U processing enzymes provides a simple safeguard.

View Article: PubMed Central - PubMed

Affiliation: Abteilung Molekulare Genetik und Präparative Molekularbiologe, Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen, Göttingen, Germany.

ABSTRACT
The mutagenic threat of hydrolytic DNA cytosine deamination is met mostly by uracil DNA glycosylases (UDG) initiating base excision repair. However, several sequenced genomes of archaeal organisms are devoid of genes coding for homologues of the otherwise ubiquitous UDG superfamily of proteins. Previously, two possible solutions to this problem were offered by (i) a report of a newly discovered family of uracil DNA glycosylases exemplified by MJ1434, a protein found in the hyperthermophilic archaeon Methanocaldococcus jannaschii, and (ii) the description of TTC0482, an EndoIV homologue from the hyperthermophilic bacterium Thermus thermophilus HB27, as being able to excise uracil from DNA. Sequence homologues of both proteins can be found throughout the archaeal domain of life. Three proteins orthologous to MJ1434 and the family founder itself were tested for but failed to exhibit DNA uracil glycosylase activity when produced in an Ung-deficient Escherichia coli host. Likewise, no DNA uracil processing activity could be detected to be associated with TTC0482, while the protein was fully active as an AP endonuclease. We propose that the uracil processing activities formerly found were due to contaminations with Ung enzyme. Use of Deltaung-strains as hosts for production of putatively DNA-U processing enzymes provides a simple safeguard.

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DNA-U glycosylase and AP lyase assays with E132K mutant of MJ1434. (A) SDS–PAGE analysis of IMAC purified MJ1434 (wild type) and MJ1434/E132K (for details refer to ‘Materials and Methods’ section). M, molecular weight markers as in Figure 2A. Note that at this state of purification impurities are detectable in the protein preparations. (B) Schematic drawing of the double-stranded U/T mismatched DNA substrate (labels as in Figure 3D). (C) Track recording of the ALF DNA sequencer after incubation of the substrate (0.12 pmol) with 2 µg of MJ1434 (wild type) or mutant E132K or/and 0.6 pmol of E. coli Ung. NaOH: with or without NaOH post-treatment. Samples containing MJ1434 or MJ1434/E132K, if not treated with NaOH, were incubated with Proteinase K prior to gel electrophoresis. For reaction details refer to ‘Materials and Methods’ section.
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Figure 4: DNA-U glycosylase and AP lyase assays with E132K mutant of MJ1434. (A) SDS–PAGE analysis of IMAC purified MJ1434 (wild type) and MJ1434/E132K (for details refer to ‘Materials and Methods’ section). M, molecular weight markers as in Figure 2A. Note that at this state of purification impurities are detectable in the protein preparations. (B) Schematic drawing of the double-stranded U/T mismatched DNA substrate (labels as in Figure 3D). (C) Track recording of the ALF DNA sequencer after incubation of the substrate (0.12 pmol) with 2 µg of MJ1434 (wild type) or mutant E132K or/and 0.6 pmol of E. coli Ung. NaOH: with or without NaOH post-treatment. Samples containing MJ1434 or MJ1434/E132K, if not treated with NaOH, were incubated with Proteinase K prior to gel electrophoresis. For reaction details refer to ‘Materials and Methods’ section.

Mentions: A mutant gene coding for MJ1434/E132K was constructed and the protein purified (by IMAC) in parallel with wild-type MJ1434 from extracts of the same Δung expression host as used before (Figure 4A). Results of DNA uracil glycosylase and AP lyase assays on these protein preparations are displayed in Figure 4C. The necessary substrate containing an AP site was produced from 2′-deoxy-oligonucleotide containing a uracil residue (Figure 4B) by treatment with uracil DNA glycosylase Ung from E. coli.Figure 4.


Helix-hairpin-helix protein MJ1434 from Methanocaldococcus jannaschii and EndoIV homologue TTC0482 from Thermus thermophilus HB27 do not process DNA uracil residues.

Schomacher L, Smolorz S, Ciirdaeva E, Ber S, Kramer W, Fritz HJ - Nucleic Acids Res. (2010)

DNA-U glycosylase and AP lyase assays with E132K mutant of MJ1434. (A) SDS–PAGE analysis of IMAC purified MJ1434 (wild type) and MJ1434/E132K (for details refer to ‘Materials and Methods’ section). M, molecular weight markers as in Figure 2A. Note that at this state of purification impurities are detectable in the protein preparations. (B) Schematic drawing of the double-stranded U/T mismatched DNA substrate (labels as in Figure 3D). (C) Track recording of the ALF DNA sequencer after incubation of the substrate (0.12 pmol) with 2 µg of MJ1434 (wild type) or mutant E132K or/and 0.6 pmol of E. coli Ung. NaOH: with or without NaOH post-treatment. Samples containing MJ1434 or MJ1434/E132K, if not treated with NaOH, were incubated with Proteinase K prior to gel electrophoresis. For reaction details refer to ‘Materials and Methods’ section.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 4: DNA-U glycosylase and AP lyase assays with E132K mutant of MJ1434. (A) SDS–PAGE analysis of IMAC purified MJ1434 (wild type) and MJ1434/E132K (for details refer to ‘Materials and Methods’ section). M, molecular weight markers as in Figure 2A. Note that at this state of purification impurities are detectable in the protein preparations. (B) Schematic drawing of the double-stranded U/T mismatched DNA substrate (labels as in Figure 3D). (C) Track recording of the ALF DNA sequencer after incubation of the substrate (0.12 pmol) with 2 µg of MJ1434 (wild type) or mutant E132K or/and 0.6 pmol of E. coli Ung. NaOH: with or without NaOH post-treatment. Samples containing MJ1434 or MJ1434/E132K, if not treated with NaOH, were incubated with Proteinase K prior to gel electrophoresis. For reaction details refer to ‘Materials and Methods’ section.
Mentions: A mutant gene coding for MJ1434/E132K was constructed and the protein purified (by IMAC) in parallel with wild-type MJ1434 from extracts of the same Δung expression host as used before (Figure 4A). Results of DNA uracil glycosylase and AP lyase assays on these protein preparations are displayed in Figure 4C. The necessary substrate containing an AP site was produced from 2′-deoxy-oligonucleotide containing a uracil residue (Figure 4B) by treatment with uracil DNA glycosylase Ung from E. coli.Figure 4.

Bottom Line: Sequence homologues of both proteins can be found throughout the archaeal domain of life.We propose that the uracil processing activities formerly found were due to contaminations with Ung enzyme.Use of Deltaung-strains as hosts for production of putatively DNA-U processing enzymes provides a simple safeguard.

View Article: PubMed Central - PubMed

Affiliation: Abteilung Molekulare Genetik und Präparative Molekularbiologe, Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen, Göttingen, Germany.

ABSTRACT
The mutagenic threat of hydrolytic DNA cytosine deamination is met mostly by uracil DNA glycosylases (UDG) initiating base excision repair. However, several sequenced genomes of archaeal organisms are devoid of genes coding for homologues of the otherwise ubiquitous UDG superfamily of proteins. Previously, two possible solutions to this problem were offered by (i) a report of a newly discovered family of uracil DNA glycosylases exemplified by MJ1434, a protein found in the hyperthermophilic archaeon Methanocaldococcus jannaschii, and (ii) the description of TTC0482, an EndoIV homologue from the hyperthermophilic bacterium Thermus thermophilus HB27, as being able to excise uracil from DNA. Sequence homologues of both proteins can be found throughout the archaeal domain of life. Three proteins orthologous to MJ1434 and the family founder itself were tested for but failed to exhibit DNA uracil glycosylase activity when produced in an Ung-deficient Escherichia coli host. Likewise, no DNA uracil processing activity could be detected to be associated with TTC0482, while the protein was fully active as an AP endonuclease. We propose that the uracil processing activities formerly found were due to contaminations with Ung enzyme. Use of Deltaung-strains as hosts for production of putatively DNA-U processing enzymes provides a simple safeguard.

Show MeSH
Related in: MedlinePlus