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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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An unusual sequence insertion is present in the DNA glycosylase domain of members of the DML family. (A) Schematic diagram showing ROS1 regions conserved among DML proteins. (B) Multiple sequence alignment of DML proteins and several HhH-GPD superfamily members. Listed above the primary sequence are indicated secondary structure assignments from the ROS1 model prediction shown in (C), colored according to regions shown in (A). The helix–hairpin–helix of the HhH-GPD motif is shown in cyan. ROS1 amino acids mutated in this study are indicated by inverted triangles and highlighted in green (Q584 and W1012), blue (F589 and Y1028), yellow (T606 and D611) or red (Q607 and N608). The lysine residue that is diagnostic of bifunctional glycosylase/lyase activity, and the conserved aspartic acid residue in the active site are indicated by asterisks. The HhH-GPD and the [4Fe–4S] cluster loop (FCL) motifs are boxed. Names of organisms are abbreviated as follows: Ath, Arabidopsis thaliana; Nta, Nicotiana tabacum; Bst, Bacillus stearothermophilus; Eco, Escherichia coli; Mth, Methanobacterium thermoautotrophicum; Mmu, Mus musculus; Hsa, Homo sapiens. Genbank accession numbers are: Ath ROS1: AAP37178; Ath DME: ABC61677; Nta ROS1: BAF52855; Bst EndoIII: 1P59; Eco EndoIII: P20625; Mth Mig: NP_039762; Eco MutY: NP_417436; Mmu MBD4: 1NGN; Hsa OGG1: O15527; Eco AlkA: P04395. (C) Ribbon diagrams of the structural model for the DNA glycosylase domain of ROS1 and the crystallographic Bst EndoIII structure used as template. Structural elements are colored as in (A). The duplex DNA is shown in orange. Nucleic acid coordinates extracted from the Bst EndoIII-DNA trapped complex were used to superimpose a DNA structure with a flipped-out AP site analog onto the ROS1 model. (D) Close-up view of the ROS1 model. Mutated residues are shown as sticks and colored according to (B). The conserved lysine and aspartic acid residues are shown in magenta.
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Figure 1: An unusual sequence insertion is present in the DNA glycosylase domain of members of the DML family. (A) Schematic diagram showing ROS1 regions conserved among DML proteins. (B) Multiple sequence alignment of DML proteins and several HhH-GPD superfamily members. Listed above the primary sequence are indicated secondary structure assignments from the ROS1 model prediction shown in (C), colored according to regions shown in (A). The helix–hairpin–helix of the HhH-GPD motif is shown in cyan. ROS1 amino acids mutated in this study are indicated by inverted triangles and highlighted in green (Q584 and W1012), blue (F589 and Y1028), yellow (T606 and D611) or red (Q607 and N608). The lysine residue that is diagnostic of bifunctional glycosylase/lyase activity, and the conserved aspartic acid residue in the active site are indicated by asterisks. The HhH-GPD and the [4Fe–4S] cluster loop (FCL) motifs are boxed. Names of organisms are abbreviated as follows: Ath, Arabidopsis thaliana; Nta, Nicotiana tabacum; Bst, Bacillus stearothermophilus; Eco, Escherichia coli; Mth, Methanobacterium thermoautotrophicum; Mmu, Mus musculus; Hsa, Homo sapiens. Genbank accession numbers are: Ath ROS1: AAP37178; Ath DME: ABC61677; Nta ROS1: BAF52855; Bst EndoIII: 1P59; Eco EndoIII: P20625; Mth Mig: NP_039762; Eco MutY: NP_417436; Mmu MBD4: 1NGN; Hsa OGG1: O15527; Eco AlkA: P04395. (C) Ribbon diagrams of the structural model for the DNA glycosylase domain of ROS1 and the crystallographic Bst EndoIII structure used as template. Structural elements are colored as in (A). The duplex DNA is shown in orange. Nucleic acid coordinates extracted from the Bst EndoIII-DNA trapped complex were used to superimpose a DNA structure with a flipped-out AP site analog onto the ROS1 model. (D) Close-up view of the ROS1 model. Mutated residues are shown as sticks and colored according to (B). The conserved lysine and aspartic acid residues are shown in magenta.

Mentions: To gain insight into residues that comprise the DNA glycosylase domain of DML proteins, we performed a multiple sequence alignment that included Arabidopsis ROS1 and DME, Nicotiana tabacum ROS1, and several HhH-GPD proteins (Figure 1). The alignment revealed that sequence similarity to HhH-GPD enzymes in DML proteins is actually distributed over two non-contiguous segments. The first segment corresponds to a region that in HhH-GPD members contains the base-flipping wedge and its flanking alfa-helixes, as well as some of the residues that line the active site pocket (32). The second segment includes the HhH-GPD motif and its invariant aspartate, which is absolutely required for catalysis of 5-meC excision by DML proteins (10,11,13,14). This region also contains a lysine residue that is only present in the subset of HhH-GPD proteins with bifunctional DNA glycosylase/lyase activity (19) and a [4Fe–4S] cluster loop (FCL) motif that in some HHh-GPD proteins, such as E. coli Endo III and MutY, ligates a [4Fe–4S] cluster (19) (Figure 1B). The two separate segments with sequence similarity to HhH-GPD proteins are interconnected by a non-conserved linker region that is highly variable in sequence and length among members of the DML family (Figure 1B and Supplementary Figure S1). We applied a well-characterized disorder predictor [VL3H (28)] to analyze the location of ordered and disordered regions in ROS1 (Supplementary Figure S4). The results predict a high disorder content within this linker region, thus suggesting that it is intrinsically unstructured under native conditions.Figure 1.


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)

An unusual sequence insertion is present in the DNA glycosylase domain of members of the DML family. (A) Schematic diagram showing ROS1 regions conserved among DML proteins. (B) Multiple sequence alignment of DML proteins and several HhH-GPD superfamily members. Listed above the primary sequence are indicated secondary structure assignments from the ROS1 model prediction shown in (C), colored according to regions shown in (A). The helix–hairpin–helix of the HhH-GPD motif is shown in cyan. ROS1 amino acids mutated in this study are indicated by inverted triangles and highlighted in green (Q584 and W1012), blue (F589 and Y1028), yellow (T606 and D611) or red (Q607 and N608). The lysine residue that is diagnostic of bifunctional glycosylase/lyase activity, and the conserved aspartic acid residue in the active site are indicated by asterisks. The HhH-GPD and the [4Fe–4S] cluster loop (FCL) motifs are boxed. Names of organisms are abbreviated as follows: Ath, Arabidopsis thaliana; Nta, Nicotiana tabacum; Bst, Bacillus stearothermophilus; Eco, Escherichia coli; Mth, Methanobacterium thermoautotrophicum; Mmu, Mus musculus; Hsa, Homo sapiens. Genbank accession numbers are: Ath ROS1: AAP37178; Ath DME: ABC61677; Nta ROS1: BAF52855; Bst EndoIII: 1P59; Eco EndoIII: P20625; Mth Mig: NP_039762; Eco MutY: NP_417436; Mmu MBD4: 1NGN; Hsa OGG1: O15527; Eco AlkA: P04395. (C) Ribbon diagrams of the structural model for the DNA glycosylase domain of ROS1 and the crystallographic Bst EndoIII structure used as template. Structural elements are colored as in (A). The duplex DNA is shown in orange. Nucleic acid coordinates extracted from the Bst EndoIII-DNA trapped complex were used to superimpose a DNA structure with a flipped-out AP site analog onto the ROS1 model. (D) Close-up view of the ROS1 model. Mutated residues are shown as sticks and colored according to (B). The conserved lysine and aspartic acid residues are shown in magenta.
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Related In: Results  -  Collection

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Figure 1: An unusual sequence insertion is present in the DNA glycosylase domain of members of the DML family. (A) Schematic diagram showing ROS1 regions conserved among DML proteins. (B) Multiple sequence alignment of DML proteins and several HhH-GPD superfamily members. Listed above the primary sequence are indicated secondary structure assignments from the ROS1 model prediction shown in (C), colored according to regions shown in (A). The helix–hairpin–helix of the HhH-GPD motif is shown in cyan. ROS1 amino acids mutated in this study are indicated by inverted triangles and highlighted in green (Q584 and W1012), blue (F589 and Y1028), yellow (T606 and D611) or red (Q607 and N608). The lysine residue that is diagnostic of bifunctional glycosylase/lyase activity, and the conserved aspartic acid residue in the active site are indicated by asterisks. The HhH-GPD and the [4Fe–4S] cluster loop (FCL) motifs are boxed. Names of organisms are abbreviated as follows: Ath, Arabidopsis thaliana; Nta, Nicotiana tabacum; Bst, Bacillus stearothermophilus; Eco, Escherichia coli; Mth, Methanobacterium thermoautotrophicum; Mmu, Mus musculus; Hsa, Homo sapiens. Genbank accession numbers are: Ath ROS1: AAP37178; Ath DME: ABC61677; Nta ROS1: BAF52855; Bst EndoIII: 1P59; Eco EndoIII: P20625; Mth Mig: NP_039762; Eco MutY: NP_417436; Mmu MBD4: 1NGN; Hsa OGG1: O15527; Eco AlkA: P04395. (C) Ribbon diagrams of the structural model for the DNA glycosylase domain of ROS1 and the crystallographic Bst EndoIII structure used as template. Structural elements are colored as in (A). The duplex DNA is shown in orange. Nucleic acid coordinates extracted from the Bst EndoIII-DNA trapped complex were used to superimpose a DNA structure with a flipped-out AP site analog onto the ROS1 model. (D) Close-up view of the ROS1 model. Mutated residues are shown as sticks and colored according to (B). The conserved lysine and aspartic acid residues are shown in magenta.
Mentions: To gain insight into residues that comprise the DNA glycosylase domain of DML proteins, we performed a multiple sequence alignment that included Arabidopsis ROS1 and DME, Nicotiana tabacum ROS1, and several HhH-GPD proteins (Figure 1). The alignment revealed that sequence similarity to HhH-GPD enzymes in DML proteins is actually distributed over two non-contiguous segments. The first segment corresponds to a region that in HhH-GPD members contains the base-flipping wedge and its flanking alfa-helixes, as well as some of the residues that line the active site pocket (32). The second segment includes the HhH-GPD motif and its invariant aspartate, which is absolutely required for catalysis of 5-meC excision by DML proteins (10,11,13,14). This region also contains a lysine residue that is only present in the subset of HhH-GPD proteins with bifunctional DNA glycosylase/lyase activity (19) and a [4Fe–4S] cluster loop (FCL) motif that in some HHh-GPD proteins, such as E. coli Endo III and MutY, ligates a [4Fe–4S] cluster (19) (Figure 1B). The two separate segments with sequence similarity to HhH-GPD proteins are interconnected by a non-conserved linker region that is highly variable in sequence and length among members of the DML family (Figure 1B and Supplementary Figure S1). We applied a well-characterized disorder predictor [VL3H (28)] to analyze the location of ordered and disordered regions in ROS1 (Supplementary Figure S4). The results predict a high disorder content within this linker region, thus suggesting that it is intrinsically unstructured under native conditions.Figure 1.

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