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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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Assays with TTC0482 for uracil processing and AP endonuclease activities. (A) SDS–PAGE analysis of purified TTC0482 and TTUDGA from T. thermophilus HB27 after IMAC and heparin affinity chromatography (for details refer to ‘Materials and Methods’ section). M, molecular weight markers as in Figure 2A. Calculated relative molecular weights for TTC0482 and TTUDGA are 31 200 and 23 680, respectively. (B) Gel electophoretic analysis of reaction products after incubation of substrates (0.12 pmol) with 0.6 pmol of the EndoIV homologue TTC0482 or/and the uracil DNA glycosylase TTUDGA (12) from T. thermophilus HB27 as indicated. The substrates are identical to the ones used for the glycosylase assay of MJ1434 (Figure 3D). NaOH±: with or without post-reaction NaOH-treatment. Note that the AP-endonuclease reaction product shown in lane 7 migrates as a 15-mer with a 3′-OH terminus (compare marker in lane 1). For reaction details refer to ‘Materials and Methods’ section.
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Figure 5: Assays with TTC0482 for uracil processing and AP endonuclease activities. (A) SDS–PAGE analysis of purified TTC0482 and TTUDGA from T. thermophilus HB27 after IMAC and heparin affinity chromatography (for details refer to ‘Materials and Methods’ section). M, molecular weight markers as in Figure 2A. Calculated relative molecular weights for TTC0482 and TTUDGA are 31 200 and 23 680, respectively. (B) Gel electophoretic analysis of reaction products after incubation of substrates (0.12 pmol) with 0.6 pmol of the EndoIV homologue TTC0482 or/and the uracil DNA glycosylase TTUDGA (12) from T. thermophilus HB27 as indicated. The substrates are identical to the ones used for the glycosylase assay of MJ1434 (Figure 3D). NaOH±: with or without post-reaction NaOH-treatment. Note that the AP-endonuclease reaction product shown in lane 7 migrates as a 15-mer with a 3′-OH terminus (compare marker in lane 1). For reaction details refer to ‘Materials and Methods’ section.

Mentions: A second candidate for the function of an initiator of DNA-U repair in M. thermautotrophicus ΔH was suggested by work of Back et al. in 2006 (25). These authors characterized the EndoIV homologue from T. thermophilus HB27, TTC0482, as an enzyme capable of processing DNA-U residues in addition to its activity as an AP endonuclease (25). The corresponding homologue in M. thermautotrophicus ΔH is MTH1010. With the hindsight, however, of our experience with MJ1434 and some of its homologues, including MTH746, we decided to first reproduce the findings of Back et al. (25) before embarking on any enzymological characterization of EndoIV homologues from M. thermautotrophicus ΔH or yet other organisms. To this end, we constructed a pET-28a-ttc0482 expression plasmid, produced the protein in BL21_UXX (30) and purified it to near homogeneity by IMAC and heparin affinity chromatography (Figure 5A).Figure 5.


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)

Assays with TTC0482 for uracil processing and AP endonuclease activities. (A) SDS–PAGE analysis of purified TTC0482 and TTUDGA from T. thermophilus HB27 after IMAC and heparin affinity chromatography (for details refer to ‘Materials and Methods’ section). M, molecular weight markers as in Figure 2A. Calculated relative molecular weights for TTC0482 and TTUDGA are 31 200 and 23 680, respectively. (B) Gel electophoretic analysis of reaction products after incubation of substrates (0.12 pmol) with 0.6 pmol of the EndoIV homologue TTC0482 or/and the uracil DNA glycosylase TTUDGA (12) from T. thermophilus HB27 as indicated. The substrates are identical to the ones used for the glycosylase assay of MJ1434 (Figure 3D). NaOH±: with or without post-reaction NaOH-treatment. Note that the AP-endonuclease reaction product shown in lane 7 migrates as a 15-mer with a 3′-OH terminus (compare marker in lane 1). For reaction details refer to ‘Materials and Methods’ section.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 5: Assays with TTC0482 for uracil processing and AP endonuclease activities. (A) SDS–PAGE analysis of purified TTC0482 and TTUDGA from T. thermophilus HB27 after IMAC and heparin affinity chromatography (for details refer to ‘Materials and Methods’ section). M, molecular weight markers as in Figure 2A. Calculated relative molecular weights for TTC0482 and TTUDGA are 31 200 and 23 680, respectively. (B) Gel electophoretic analysis of reaction products after incubation of substrates (0.12 pmol) with 0.6 pmol of the EndoIV homologue TTC0482 or/and the uracil DNA glycosylase TTUDGA (12) from T. thermophilus HB27 as indicated. The substrates are identical to the ones used for the glycosylase assay of MJ1434 (Figure 3D). NaOH±: with or without post-reaction NaOH-treatment. Note that the AP-endonuclease reaction product shown in lane 7 migrates as a 15-mer with a 3′-OH terminus (compare marker in lane 1). For reaction details refer to ‘Materials and Methods’ section.
Mentions: A second candidate for the function of an initiator of DNA-U repair in M. thermautotrophicus ΔH was suggested by work of Back et al. in 2006 (25). These authors characterized the EndoIV homologue from T. thermophilus HB27, TTC0482, as an enzyme capable of processing DNA-U residues in addition to its activity as an AP endonuclease (25). The corresponding homologue in M. thermautotrophicus ΔH is MTH1010. With the hindsight, however, of our experience with MJ1434 and some of its homologues, including MTH746, we decided to first reproduce the findings of Back et al. (25) before embarking on any enzymological characterization of EndoIV homologues from M. thermautotrophicus ΔH or yet other organisms. To this end, we constructed a pET-28a-ttc0482 expression plasmid, produced the protein in BL21_UXX (30) and purified it to near homogeneity by IMAC and heparin affinity chromatography (Figure 5A).Figure 5.

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