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Uracil-DNA glycosylases SMUG1 and UNG2 coordinate the initial steps of base excision repair by distinct mechanisms.

Pettersen HS, Sundheim O, Gilljam KM, Slupphaug G, Krokan HE, Kavli B - Nucleic Acids Res. (2007)

Bottom Line: Mutations in this motif increase catalytic turnover due to reduced product binding.In contrast, the highly efficient UNG2 lacks product-binding capacity and stimulates AP-site cleavage by APE1, facilitating the two first steps in BER.In summary, this work reveals that SMUG1 and UNG2 coordinate the initial steps of BER by distinct mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Research and Molecular Medicine, NTNU, N-7006 Trondheim, Norway.

ABSTRACT
DNA glycosylases UNG and SMUG1 excise uracil from DNA and belong to the same protein superfamily. Vertebrates contain both SMUG1 and UNG, but their distinct roles in base excision repair (BER) of deaminated cytosine (U:G) are still not fully defined. Here we have examined the ability of human SMUG1 and UNG2 (nuclear UNG) to initiate and coordinate repair of U:G mismatches. When expressed in Escherichia coli cells, human UNG2 initiates complete repair of deaminated cytosine, while SMUG1 inhibits cell proliferation. In vitro, we show that SMUG1 binds tightly to AP-sites and inhibits AP-site cleavage by AP-endonucleases. Furthermore, a specific motif important for the AP-site product binding has been identified. Mutations in this motif increase catalytic turnover due to reduced product binding. In contrast, the highly efficient UNG2 lacks product-binding capacity and stimulates AP-site cleavage by APE1, facilitating the two first steps in BER. In summary, this work reveals that SMUG1 and UNG2 coordinate the initial steps of BER by distinct mechanisms. UNG2 is apparently adapted to rapid and highly coordinated repair of uracil (U:G and U:A) in replicating DNA, while the less efficient SMUG1 may be more important in repair of deaminated cytosine (U:G) in non-replicating chromatin.

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Alignment of SMUG1 orthologs arranged in descending order of sequence similarity to hSMUG1. Sequences were obtained by TBLASTN 2.2.14 including GenBank, EMBL, DDBJ and PDB sequences (54). The alignment of the SMUG1 orthologs was generated using ClustalW (55); the final alignment was made with Jalview (56). Secondary structure of xSMUG1 is illustrated above the alignment. The alignment displays the important functional motifs characterizing the SMUG1 enzyme with its description above. Individual residues within each motif are coloured according to ClustalX colour coding. Species and accession number used are as follows: Hs (H. sapiens, AAL86910.1), Pt (Pan troglodytes, XP_509109), Mm (M. musculus, Q6P5C5), Bt (Bos Taurus, Q59I47), Rn (Rattus norvegicus, Q811Q1), Cf (Canis familiaris, XP_543623.2), Tn (Tetraodon nigroviridis, CAF95523.1), Xl (Xenopus laevis, Q9YGN6), Gm (Geobacter metallireducens GS-15, YP_282069.1), AE (Azoarcus sp. EbN1, YP_158606.1),Ci (Ciona intestinalis, AK115076.1), Sp (Strongylocentrotus purpuratus, XP_782746.1), Rb (Rhodopirellula baltica SH 1, NP_869403.1), Tc (Tribolium castaneum, XP_971699.1), Ag (Anopheles gambiae str. PEST, XP_312038.2), Am (Apis mellifera, XP_396883.2), Dm (Drosophila melanogaster, NP_650609.1), Dp (Drosophila pseudoobscura, EAL272349.1).
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Figure 1: Alignment of SMUG1 orthologs arranged in descending order of sequence similarity to hSMUG1. Sequences were obtained by TBLASTN 2.2.14 including GenBank, EMBL, DDBJ and PDB sequences (54). The alignment of the SMUG1 orthologs was generated using ClustalW (55); the final alignment was made with Jalview (56). Secondary structure of xSMUG1 is illustrated above the alignment. The alignment displays the important functional motifs characterizing the SMUG1 enzyme with its description above. Individual residues within each motif are coloured according to ClustalX colour coding. Species and accession number used are as follows: Hs (H. sapiens, AAL86910.1), Pt (Pan troglodytes, XP_509109), Mm (M. musculus, Q6P5C5), Bt (Bos Taurus, Q59I47), Rn (Rattus norvegicus, Q811Q1), Cf (Canis familiaris, XP_543623.2), Tn (Tetraodon nigroviridis, CAF95523.1), Xl (Xenopus laevis, Q9YGN6), Gm (Geobacter metallireducens GS-15, YP_282069.1), AE (Azoarcus sp. EbN1, YP_158606.1),Ci (Ciona intestinalis, AK115076.1), Sp (Strongylocentrotus purpuratus, XP_782746.1), Rb (Rhodopirellula baltica SH 1, NP_869403.1), Tc (Tribolium castaneum, XP_971699.1), Ag (Anopheles gambiae str. PEST, XP_312038.2), Am (Apis mellifera, XP_396883.2), Dm (Drosophila melanogaster, NP_650609.1), Dp (Drosophila pseudoobscura, EAL272349.1).

Mentions: SMUG1 was suggested to be a relatively new evolutionary offspring in the UDG superfamily found only in vertebrates and insects (6,17). However, a BLAST search using the sequence of hSMUG1 protein as query revealed SMUG1 orthologs both in prokaryotes (proteobacteria and planctomycetes) and in marine non-vertebrates such as sea urchin and sea squirt (Figure 1). Remarkably, the vertebrate SMUG1 has highest similarity to sequences identified in bacteria, showing 51.1% identity and 69.9% similarity between human SMUG1 and SMUG1 from Geobacter metallireducens (Figure 1). We did not find UNG genes in the SMUG1-containing non-vertebrate organisms identified here except in sea urchin, neither is it present in insects. Moreover, the prokaryotes encoding SMUG1 also lack orthologs of other members of the UDG family [MUG, UDG 4 (25) and UDG 5 (26)], indicating that SMUG1 may be the only uracil-DNA glycosylase in these species.Figure 1.


Uracil-DNA glycosylases SMUG1 and UNG2 coordinate the initial steps of base excision repair by distinct mechanisms.

Pettersen HS, Sundheim O, Gilljam KM, Slupphaug G, Krokan HE, Kavli B - Nucleic Acids Res. (2007)

Alignment of SMUG1 orthologs arranged in descending order of sequence similarity to hSMUG1. Sequences were obtained by TBLASTN 2.2.14 including GenBank, EMBL, DDBJ and PDB sequences (54). The alignment of the SMUG1 orthologs was generated using ClustalW (55); the final alignment was made with Jalview (56). Secondary structure of xSMUG1 is illustrated above the alignment. The alignment displays the important functional motifs characterizing the SMUG1 enzyme with its description above. Individual residues within each motif are coloured according to ClustalX colour coding. Species and accession number used are as follows: Hs (H. sapiens, AAL86910.1), Pt (Pan troglodytes, XP_509109), Mm (M. musculus, Q6P5C5), Bt (Bos Taurus, Q59I47), Rn (Rattus norvegicus, Q811Q1), Cf (Canis familiaris, XP_543623.2), Tn (Tetraodon nigroviridis, CAF95523.1), Xl (Xenopus laevis, Q9YGN6), Gm (Geobacter metallireducens GS-15, YP_282069.1), AE (Azoarcus sp. EbN1, YP_158606.1),Ci (Ciona intestinalis, AK115076.1), Sp (Strongylocentrotus purpuratus, XP_782746.1), Rb (Rhodopirellula baltica SH 1, NP_869403.1), Tc (Tribolium castaneum, XP_971699.1), Ag (Anopheles gambiae str. PEST, XP_312038.2), Am (Apis mellifera, XP_396883.2), Dm (Drosophila melanogaster, NP_650609.1), Dp (Drosophila pseudoobscura, EAL272349.1).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 1: Alignment of SMUG1 orthologs arranged in descending order of sequence similarity to hSMUG1. Sequences were obtained by TBLASTN 2.2.14 including GenBank, EMBL, DDBJ and PDB sequences (54). The alignment of the SMUG1 orthologs was generated using ClustalW (55); the final alignment was made with Jalview (56). Secondary structure of xSMUG1 is illustrated above the alignment. The alignment displays the important functional motifs characterizing the SMUG1 enzyme with its description above. Individual residues within each motif are coloured according to ClustalX colour coding. Species and accession number used are as follows: Hs (H. sapiens, AAL86910.1), Pt (Pan troglodytes, XP_509109), Mm (M. musculus, Q6P5C5), Bt (Bos Taurus, Q59I47), Rn (Rattus norvegicus, Q811Q1), Cf (Canis familiaris, XP_543623.2), Tn (Tetraodon nigroviridis, CAF95523.1), Xl (Xenopus laevis, Q9YGN6), Gm (Geobacter metallireducens GS-15, YP_282069.1), AE (Azoarcus sp. EbN1, YP_158606.1),Ci (Ciona intestinalis, AK115076.1), Sp (Strongylocentrotus purpuratus, XP_782746.1), Rb (Rhodopirellula baltica SH 1, NP_869403.1), Tc (Tribolium castaneum, XP_971699.1), Ag (Anopheles gambiae str. PEST, XP_312038.2), Am (Apis mellifera, XP_396883.2), Dm (Drosophila melanogaster, NP_650609.1), Dp (Drosophila pseudoobscura, EAL272349.1).
Mentions: SMUG1 was suggested to be a relatively new evolutionary offspring in the UDG superfamily found only in vertebrates and insects (6,17). However, a BLAST search using the sequence of hSMUG1 protein as query revealed SMUG1 orthologs both in prokaryotes (proteobacteria and planctomycetes) and in marine non-vertebrates such as sea urchin and sea squirt (Figure 1). Remarkably, the vertebrate SMUG1 has highest similarity to sequences identified in bacteria, showing 51.1% identity and 69.9% similarity between human SMUG1 and SMUG1 from Geobacter metallireducens (Figure 1). We did not find UNG genes in the SMUG1-containing non-vertebrate organisms identified here except in sea urchin, neither is it present in insects. Moreover, the prokaryotes encoding SMUG1 also lack orthologs of other members of the UDG family [MUG, UDG 4 (25) and UDG 5 (26)], indicating that SMUG1 may be the only uracil-DNA glycosylase in these species.Figure 1.

Bottom Line: Mutations in this motif increase catalytic turnover due to reduced product binding.In contrast, the highly efficient UNG2 lacks product-binding capacity and stimulates AP-site cleavage by APE1, facilitating the two first steps in BER.In summary, this work reveals that SMUG1 and UNG2 coordinate the initial steps of BER by distinct mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Research and Molecular Medicine, NTNU, N-7006 Trondheim, Norway.

ABSTRACT
DNA glycosylases UNG and SMUG1 excise uracil from DNA and belong to the same protein superfamily. Vertebrates contain both SMUG1 and UNG, but their distinct roles in base excision repair (BER) of deaminated cytosine (U:G) are still not fully defined. Here we have examined the ability of human SMUG1 and UNG2 (nuclear UNG) to initiate and coordinate repair of U:G mismatches. When expressed in Escherichia coli cells, human UNG2 initiates complete repair of deaminated cytosine, while SMUG1 inhibits cell proliferation. In vitro, we show that SMUG1 binds tightly to AP-sites and inhibits AP-site cleavage by AP-endonucleases. Furthermore, a specific motif important for the AP-site product binding has been identified. Mutations in this motif increase catalytic turnover due to reduced product binding. In contrast, the highly efficient UNG2 lacks product-binding capacity and stimulates AP-site cleavage by APE1, facilitating the two first steps in BER. In summary, this work reveals that SMUG1 and UNG2 coordinate the initial steps of BER by distinct mechanisms. UNG2 is apparently adapted to rapid and highly coordinated repair of uracil (U:G and U:A) in replicating DNA, while the less efficient SMUG1 may be more important in repair of deaminated cytosine (U:G) in non-replicating chromatin.

Show MeSH
Related in: MedlinePlus