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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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N608 contributes to sequence-context specificity. (A) Purified WT ROS1 or mutant variants (20 nM) were incubated at 30°C for 4 h with 51-mer double-stranded oligonucleotide substrates (20 nM) containing at position 29 of the labeled upper-strand a 5-meC residue in different sequence contexts. Products were separated in a 12% denaturing polyacrylamide gel and the amount of incised oligonucleotide was quantified by fluorescent scanning. For ease of comparison, the incision values for each substrate are normalized to the total incision detected in all four substrates for each individual enzyme. (B) Substrate processing ability of type ROS1 and the mutant variant N608 in different sequence contexts. Relative processing efficiencies were determined in kinetic assays as described in ‘Materials and Methods’ section. Purified proteins (20 nM), were incubated at 30°C with 51-mer double-stranded oligonucleotide substrates (20 nM) containing at position 29 of the labeled upper-strand a 5-meC residue in different sequence contexts. Reaction products were separated in a 12% denaturing polyacrylamide gel and quantified by fluorescence scanning. Values are means ± SE (error bars) from two independent experiments.
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Figure 6: N608 contributes to sequence-context specificity. (A) Purified WT ROS1 or mutant variants (20 nM) were incubated at 30°C for 4 h with 51-mer double-stranded oligonucleotide substrates (20 nM) containing at position 29 of the labeled upper-strand a 5-meC residue in different sequence contexts. Products were separated in a 12% denaturing polyacrylamide gel and the amount of incised oligonucleotide was quantified by fluorescent scanning. For ease of comparison, the incision values for each substrate are normalized to the total incision detected in all four substrates for each individual enzyme. (B) Substrate processing ability of type ROS1 and the mutant variant N608 in different sequence contexts. Relative processing efficiencies were determined in kinetic assays as described in ‘Materials and Methods’ section. Purified proteins (20 nM), were incubated at 30°C with 51-mer double-stranded oligonucleotide substrates (20 nM) containing at position 29 of the labeled upper-strand a 5-meC residue in different sequence contexts. Reaction products were separated in a 12% denaturing polyacrylamide gel and quantified by fluorescence scanning. Values are means ± SE (error bars) from two independent experiments.

Mentions: In order to determine whether any of the mutated residues contributes to sequence-context specificity, we tested all ROS1 variants for their relative capacity to excise 5-meC from CG, CHG and CHH sequences (Figure 6A). We found that all mutants except N608A exhibited a sequence-context specificity similar to that of WT ROS1. Unlike the WT enzyme and the rest of ROS1 variants, the N608A mutant showed a significantly reduced activity on the asymmetric CHH context (Figure 6A). To confirm this result, we performed a kinetic analysis to compare the relative processing efficiencies of WT ROS1 and N608A on 5-meC located in CG, CHG or CHH contexts (Figure 6B). We found that both enzymes displayed a similar efficiency on CG sites, and also showed a very low activity on the CHG context when H = C. However, the N608A mutation caused a higher efficiency than WT ROS1 on CAG sequences, and a strongly reduced activity on the asymmetric CHH context. These results indicate that N608 contributes to the sequence context specificity of ROS1, and suggest that this residue may contact the DNA bases surrounding the 5-meC.Figure 6.


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)

N608 contributes to sequence-context specificity. (A) Purified WT ROS1 or mutant variants (20 nM) were incubated at 30°C for 4 h with 51-mer double-stranded oligonucleotide substrates (20 nM) containing at position 29 of the labeled upper-strand a 5-meC residue in different sequence contexts. Products were separated in a 12% denaturing polyacrylamide gel and the amount of incised oligonucleotide was quantified by fluorescent scanning. For ease of comparison, the incision values for each substrate are normalized to the total incision detected in all four substrates for each individual enzyme. (B) Substrate processing ability of type ROS1 and the mutant variant N608 in different sequence contexts. Relative processing efficiencies were determined in kinetic assays as described in ‘Materials and Methods’ section. Purified proteins (20 nM), were incubated at 30°C with 51-mer double-stranded oligonucleotide substrates (20 nM) containing at position 29 of the labeled upper-strand a 5-meC residue in different sequence contexts. Reaction products were separated in a 12% denaturing polyacrylamide gel and quantified by fluorescence scanning. Values are means ± SE (error bars) from two independent experiments.
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Figure 6: N608 contributes to sequence-context specificity. (A) Purified WT ROS1 or mutant variants (20 nM) were incubated at 30°C for 4 h with 51-mer double-stranded oligonucleotide substrates (20 nM) containing at position 29 of the labeled upper-strand a 5-meC residue in different sequence contexts. Products were separated in a 12% denaturing polyacrylamide gel and the amount of incised oligonucleotide was quantified by fluorescent scanning. For ease of comparison, the incision values for each substrate are normalized to the total incision detected in all four substrates for each individual enzyme. (B) Substrate processing ability of type ROS1 and the mutant variant N608 in different sequence contexts. Relative processing efficiencies were determined in kinetic assays as described in ‘Materials and Methods’ section. Purified proteins (20 nM), were incubated at 30°C with 51-mer double-stranded oligonucleotide substrates (20 nM) containing at position 29 of the labeled upper-strand a 5-meC residue in different sequence contexts. Reaction products were separated in a 12% denaturing polyacrylamide gel and quantified by fluorescence scanning. Values are means ± SE (error bars) from two independent experiments.
Mentions: In order to determine whether any of the mutated residues contributes to sequence-context specificity, we tested all ROS1 variants for their relative capacity to excise 5-meC from CG, CHG and CHH sequences (Figure 6A). We found that all mutants except N608A exhibited a sequence-context specificity similar to that of WT ROS1. Unlike the WT enzyme and the rest of ROS1 variants, the N608A mutant showed a significantly reduced activity on the asymmetric CHH context (Figure 6A). To confirm this result, we performed a kinetic analysis to compare the relative processing efficiencies of WT ROS1 and N608A on 5-meC located in CG, CHG or CHH contexts (Figure 6B). We found that both enzymes displayed a similar efficiency on CG sites, and also showed a very low activity on the CHG context when H = C. However, the N608A mutation caused a higher efficiency than WT ROS1 on CAG sequences, and a strongly reduced activity on the asymmetric CHH context. These results indicate that N608 contributes to the sequence context specificity of ROS1, and suggest that this residue may contact the DNA bases surrounding the 5-meC.Figure 6.

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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