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A discontinuous DNA glycosylase domain in a family of enzymes that excise 5-methylcytosine.

Ponferrada-Marín MI, Parrilla-Doblas JT, Roldán-Arjona T, Ariza RR - Nucleic Acids Res. (2010)

Bottom Line: We have found that sequence similarity to HhH-GPD enzymes in DML proteins is actually distributed over two non-contiguous segments connected by a predicted disordered region.We found that amino acids T606 and D611 are essential for ROS1 DNA glycosylase activity, whereas mutations in either of two aromatic residues (F589 and Y1028) reverse the characteristic ROS1 preference for 5-meC over T.We also found evidence suggesting that ROS1 uses Q607 to flip out 5-meC, while the contiguous N608 residue contributes to sequence-context specificity.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Córdoba, 14071-Córdoba, Spain.

ABSTRACT
DNA cytosine methylation (5-meC) is a widespread epigenetic mark associated to gene silencing. In plants, DEMETER-LIKE (DML) proteins typified by Arabidopsis REPRESSOR OF SILENCING 1 (ROS1) initiate active DNA demethylation by catalyzing 5-meC excision. DML proteins belong to the HhH-GPD superfamily, the largest and most functionally diverse group of DNA glycosylases, but the molecular properties that underlie their capacity to specifically recognize and excise 5-meC are largely unknown. We have found that sequence similarity to HhH-GPD enzymes in DML proteins is actually distributed over two non-contiguous segments connected by a predicted disordered region. We used homology-based modeling to locate candidate residues important for ROS1 function in both segments, and tested our predictions by site-specific mutagenesis. We found that amino acids T606 and D611 are essential for ROS1 DNA glycosylase activity, whereas mutations in either of two aromatic residues (F589 and Y1028) reverse the characteristic ROS1 preference for 5-meC over T. We also found evidence suggesting that ROS1 uses Q607 to flip out 5-meC, while the contiguous N608 residue contributes to sequence-context specificity. In addition to providing novel insights into the molecular basis of 5-meC excision, our results reveal that ROS1 and its DML homologs possess a discontinuous catalytic domain that is unprecedented among known DNA glycosylases.

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T606 and D611 are essential for ROS1 DNA glycosylase activity. (A) The generation of incision products was measured by incubating purified WT ROS1 or mutant variants (20 nM) at 30°C for 2 h with a double-stranded oligonucleotide substrate (20 nM) containing a single 5-meC:G pair. Samples were treated with or without NaOH 100 mM, and immediately transferred to 90°C for 10 min. Products were separated in a 12% denaturing polyacrylamide gel and the amounts of incised oligonucleotide were quantified by fluorescent scanning. (B) Purified WT ROS1 or mutant variants (20 nM) were incubated at 30°C for 2 h with a double-stranded oligonucleotide substrate (20 nM) containing a single 5-meC:G pair, either in the absence or the presence of human APE I (5 U), as indicated. Products were separated in a 12% denaturing polyacrylamide gel and the incised products were detected by fluorescent scanning. (C) A double-stranded oligonucleotide substrate containing an AP site opposite G (200 nM) was incubated at 30°C either in the absence of enzyme or in the presence of purified WT ROS1, T606L or D611V (100 nM). Reactions were stopped at the indicated times, products were separated in a 12% denaturing polyacrylamide gel and the amount of incised oligonucleotide was quantified by fluorescent scanning. Values are means ± SE (error bars) from two independent experiments. The asterisks indicate that the incision levels were significantly different (P < 0.05) from those observed in the absence of enzyme. The respective P-values were calculated using a Student’s unpaired t-test.
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Figure 3: T606 and D611 are essential for ROS1 DNA glycosylase activity. (A) The generation of incision products was measured by incubating purified WT ROS1 or mutant variants (20 nM) at 30°C for 2 h with a double-stranded oligonucleotide substrate (20 nM) containing a single 5-meC:G pair. Samples were treated with or without NaOH 100 mM, and immediately transferred to 90°C for 10 min. Products were separated in a 12% denaturing polyacrylamide gel and the amounts of incised oligonucleotide were quantified by fluorescent scanning. (B) Purified WT ROS1 or mutant variants (20 nM) were incubated at 30°C for 2 h with a double-stranded oligonucleotide substrate (20 nM) containing a single 5-meC:G pair, either in the absence or the presence of human APE I (5 U), as indicated. Products were separated in a 12% denaturing polyacrylamide gel and the incised products were detected by fluorescent scanning. (C) A double-stranded oligonucleotide substrate containing an AP site opposite G (200 nM) was incubated at 30°C either in the absence of enzyme or in the presence of purified WT ROS1, T606L or D611V (100 nM). Reactions were stopped at the indicated times, products were separated in a 12% denaturing polyacrylamide gel and the amount of incised oligonucleotide was quantified by fluorescent scanning. Values are means ± SE (error bars) from two independent experiments. The asterisks indicate that the incision levels were significantly different (P < 0.05) from those observed in the absence of enzyme. The respective P-values were calculated using a Student’s unpaired t-test.

Mentions: Since ROS1 is a bifunctional enzyme, we asked whether these mutant proteins lack DNA glycosylase activity, lyase activity or both. To differentiate 5-meC excision and strand incision, we analyzed the reaction products generated by different ROS1 variants with or without additional alkaline treatment with NaOH (Figure 3A). Incisions in the absence of NaOH reveal the combined DNA glycosylase/AP lyase action of the enzyme, whereas the alkaline treatment cleaves all AP sites generated by the enzyme and reflects DNA glycosylase activity. Consistent with our previously reported observations (21), we found that the amount of incision products generated by WT ROS1 was only slightly increased after NaOH treatment, thus suggesting that glycosyl bond scission is usually coupled to the AP lyase step. The same pattern was observed for all ROS1 mutant enzymes except T606L and D611V, which did not generate detectable incision products either in the absence or the presence of NaOH. The incapacity of both mutants to generate abasic sites was confirmed by performing reactions in the presence of human AP endonuclease APE1 (Figure 3B). We next tested whether T606L or D611V retained AP lyase activity by incubating both proteins with a 51-mer duplex oligo substrate that contained an AP site opposite G at position 29 in a CG context (Figure 3C). Although a significant level of spontaneous AP incision is observed in the absence of enzyme, the amounts of enzyme-dependent strand incision after 0.5, 2 and 24 h incubation were similar to those generated by WT ROS1 (Figure 3C; a representative gel is shown in Supplementary Figure S5). We also found that the incision product generated by both WT and mutant proteins migrates as a β-elimination product (Supplementary Figure S5). Altogether, these results indicate that both T606L and D611V mutants lack DNA glycosylase activity but retain AP lyase activity. In addition, they provide experimental evidence of the location of critical catalytic residues in the first segment of the discontinuous ROS1 DNA glycosylase domain.Figure 3.


A discontinuous DNA glycosylase domain in a family of enzymes that excise 5-methylcytosine.

Ponferrada-Marín MI, Parrilla-Doblas JT, Roldán-Arjona T, Ariza RR - Nucleic Acids Res. (2010)

T606 and D611 are essential for ROS1 DNA glycosylase activity. (A) The generation of incision products was measured by incubating purified WT ROS1 or mutant variants (20 nM) at 30°C for 2 h with a double-stranded oligonucleotide substrate (20 nM) containing a single 5-meC:G pair. Samples were treated with or without NaOH 100 mM, and immediately transferred to 90°C for 10 min. Products were separated in a 12% denaturing polyacrylamide gel and the amounts of incised oligonucleotide were quantified by fluorescent scanning. (B) Purified WT ROS1 or mutant variants (20 nM) were incubated at 30°C for 2 h with a double-stranded oligonucleotide substrate (20 nM) containing a single 5-meC:G pair, either in the absence or the presence of human APE I (5 U), as indicated. Products were separated in a 12% denaturing polyacrylamide gel and the incised products were detected by fluorescent scanning. (C) A double-stranded oligonucleotide substrate containing an AP site opposite G (200 nM) was incubated at 30°C either in the absence of enzyme or in the presence of purified WT ROS1, T606L or D611V (100 nM). Reactions were stopped at the indicated times, products were separated in a 12% denaturing polyacrylamide gel and the amount of incised oligonucleotide was quantified by fluorescent scanning. Values are means ± SE (error bars) from two independent experiments. The asterisks indicate that the incision levels were significantly different (P < 0.05) from those observed in the absence of enzyme. The respective P-values were calculated using a Student’s unpaired t-test.
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Figure 3: T606 and D611 are essential for ROS1 DNA glycosylase activity. (A) The generation of incision products was measured by incubating purified WT ROS1 or mutant variants (20 nM) at 30°C for 2 h with a double-stranded oligonucleotide substrate (20 nM) containing a single 5-meC:G pair. Samples were treated with or without NaOH 100 mM, and immediately transferred to 90°C for 10 min. Products were separated in a 12% denaturing polyacrylamide gel and the amounts of incised oligonucleotide were quantified by fluorescent scanning. (B) Purified WT ROS1 or mutant variants (20 nM) were incubated at 30°C for 2 h with a double-stranded oligonucleotide substrate (20 nM) containing a single 5-meC:G pair, either in the absence or the presence of human APE I (5 U), as indicated. Products were separated in a 12% denaturing polyacrylamide gel and the incised products were detected by fluorescent scanning. (C) A double-stranded oligonucleotide substrate containing an AP site opposite G (200 nM) was incubated at 30°C either in the absence of enzyme or in the presence of purified WT ROS1, T606L or D611V (100 nM). Reactions were stopped at the indicated times, products were separated in a 12% denaturing polyacrylamide gel and the amount of incised oligonucleotide was quantified by fluorescent scanning. Values are means ± SE (error bars) from two independent experiments. The asterisks indicate that the incision levels were significantly different (P < 0.05) from those observed in the absence of enzyme. The respective P-values were calculated using a Student’s unpaired t-test.
Mentions: Since ROS1 is a bifunctional enzyme, we asked whether these mutant proteins lack DNA glycosylase activity, lyase activity or both. To differentiate 5-meC excision and strand incision, we analyzed the reaction products generated by different ROS1 variants with or without additional alkaline treatment with NaOH (Figure 3A). Incisions in the absence of NaOH reveal the combined DNA glycosylase/AP lyase action of the enzyme, whereas the alkaline treatment cleaves all AP sites generated by the enzyme and reflects DNA glycosylase activity. Consistent with our previously reported observations (21), we found that the amount of incision products generated by WT ROS1 was only slightly increased after NaOH treatment, thus suggesting that glycosyl bond scission is usually coupled to the AP lyase step. The same pattern was observed for all ROS1 mutant enzymes except T606L and D611V, which did not generate detectable incision products either in the absence or the presence of NaOH. The incapacity of both mutants to generate abasic sites was confirmed by performing reactions in the presence of human AP endonuclease APE1 (Figure 3B). We next tested whether T606L or D611V retained AP lyase activity by incubating both proteins with a 51-mer duplex oligo substrate that contained an AP site opposite G at position 29 in a CG context (Figure 3C). Although a significant level of spontaneous AP incision is observed in the absence of enzyme, the amounts of enzyme-dependent strand incision after 0.5, 2 and 24 h incubation were similar to those generated by WT ROS1 (Figure 3C; a representative gel is shown in Supplementary Figure S5). We also found that the incision product generated by both WT and mutant proteins migrates as a β-elimination product (Supplementary Figure S5). Altogether, these results indicate that both T606L and D611V mutants lack DNA glycosylase activity but retain AP lyase activity. In addition, they provide experimental evidence of the location of critical catalytic residues in the first segment of the discontinuous ROS1 DNA glycosylase domain.Figure 3.

Bottom Line: We have found that sequence similarity to HhH-GPD enzymes in DML proteins is actually distributed over two non-contiguous segments connected by a predicted disordered region.We found that amino acids T606 and D611 are essential for ROS1 DNA glycosylase activity, whereas mutations in either of two aromatic residues (F589 and Y1028) reverse the characteristic ROS1 preference for 5-meC over T.We also found evidence suggesting that ROS1 uses Q607 to flip out 5-meC, while the contiguous N608 residue contributes to sequence-context specificity.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Córdoba, 14071-Córdoba, Spain.

ABSTRACT
DNA cytosine methylation (5-meC) is a widespread epigenetic mark associated to gene silencing. In plants, DEMETER-LIKE (DML) proteins typified by Arabidopsis REPRESSOR OF SILENCING 1 (ROS1) initiate active DNA demethylation by catalyzing 5-meC excision. DML proteins belong to the HhH-GPD superfamily, the largest and most functionally diverse group of DNA glycosylases, but the molecular properties that underlie their capacity to specifically recognize and excise 5-meC are largely unknown. We have found that sequence similarity to HhH-GPD enzymes in DML proteins is actually distributed over two non-contiguous segments connected by a predicted disordered region. We used homology-based modeling to locate candidate residues important for ROS1 function in both segments, and tested our predictions by site-specific mutagenesis. We found that amino acids T606 and D611 are essential for ROS1 DNA glycosylase activity, whereas mutations in either of two aromatic residues (F589 and Y1028) reverse the characteristic ROS1 preference for 5-meC over T. We also found evidence suggesting that ROS1 uses Q607 to flip out 5-meC, while the contiguous N608 residue contributes to sequence-context specificity. In addition to providing novel insights into the molecular basis of 5-meC excision, our results reveal that ROS1 and its DML homologs possess a discontinuous catalytic domain that is unprecedented among known DNA glycosylases.

Show MeSH
Related in: MedlinePlus