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Recognition and processing of double-stranded DNA by ExoX, a distributive 3'-5' exonuclease.

Wang T, Sun HL, Cheng F, Zhang XE, Bi L, Jiang T - Nucleic Acids Res. (2013)

Bottom Line: When ExoX complexes with blunt-ended dsDNA or 5' overhanging dsDNA, a 'wedge' composed of Leu12 and Gln13 penetrates between the first two base pairs to break the 3' terminal base pair and facilitates precise feeding of the 3' terminus of the substrate strand into the ExoX cleavage active site.Site-directed mutagenesis showed that the complementary strand-binding site and the wedge of ExoX are dsDNA specific.Together with the results of structural comparisons, our data support a mechanism by which normal and mismatched dsDNA are recognized and digested by E. coli ExoX.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China, Graduate School of Chinese Academy of Sciences, Beijing 100039, China and State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.

ABSTRACT
Members of the DnaQ superfamily are major 3'-5' exonucleases that degrade either only single-stranded DNA (ssDNA) or both ssDNA and double-stranded DNA (dsDNA). However, the mechanism by which dsDNA is recognized and digested remains unclear. Exonuclease X (ExoX) is a distributive DnaQ exonuclease that cleaves both ssDNA and dsDNA substrates. Here, we report the crystal structures of Escherichia coli ExoX in complex with three different dsDNA substrates: 3' overhanging dsDNA, blunt-ended dsDNA and 3' recessed mismatch-containing dsDNA. In these structures, ExoX binds to dsDNA via both a conserved substrate strand-interacting site and a previously uncharacterized complementary strand-interacting motif. When ExoX complexes with blunt-ended dsDNA or 5' overhanging dsDNA, a 'wedge' composed of Leu12 and Gln13 penetrates between the first two base pairs to break the 3' terminal base pair and facilitates precise feeding of the 3' terminus of the substrate strand into the ExoX cleavage active site. Site-directed mutagenesis showed that the complementary strand-binding site and the wedge of ExoX are dsDNA specific. Together with the results of structural comparisons, our data support a mechanism by which normal and mismatched dsDNA are recognized and digested by E. coli ExoX. The crystal structures also provide insight into the structural framework of the different substrate specificities of the DnaQ family members.

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Overall structure of complex I. (A) Ribbon diagram of ExoX in complex with 3′ overhanging dsDNA (ratio of 2:1). (B) Close-up view of the active site of ExoX; the DNA scissile strand twists and inserts into the active cavity of ExoX. Strong density peaks are observed at the two conserved metal-binding sites of the DnaQ family, suggesting the location of the two metal ions in ExoX. The difference (Fo-Fc) maps are contoured at 2.5σ.
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gkt495-F1: Overall structure of complex I. (A) Ribbon diagram of ExoX in complex with 3′ overhanging dsDNA (ratio of 2:1). (B) Close-up view of the active site of ExoX; the DNA scissile strand twists and inserts into the active cavity of ExoX. Strong density peaks are observed at the two conserved metal-binding sites of the DnaQ family, suggesting the location of the two metal ions in ExoX. The difference (Fo-Fc) maps are contoured at 2.5σ.

Mentions: Although attempts to crystallize full-length ExoX failed, we were able to crystallize and solve the crystal structure of a C-terminal truncation of ExoX containing residues 1–167 (hereafter referred to as ExoX, Supplementary Figure S1), which retained activity. In each asymmetrical unit, two protein molecules (A and B) bind to the ends of a single dsDNA molecule, thus forming a dumbbell-like structure (Figure 1A). Each monomer of ExoX forms a bean-shaped core consisting of a central β-sheet (strands β1–β5) surrounded by eight α-helices (α1–α8), with helices α3–α4 on one side and helices α1–α2 and α5–α8 on the other side (Figure 1A). Analysis of the ExoX structure using the Dali server showed that ExoX has the highest degree of similarity to the ε subunit of DNA polymerase III (Z-score of 18.6, PDB code: 1J53). Despite these proteins sharing only ∼33% sequence identity, ExoX can be superimposed on the ε subunit of DNA polymerase III with a root-mean-square deviation for the Cα atoms of only 1.9 Å. This similarity, together with the presence of all three Exo motifs in ExoX (Supplementary Figure S2), provided further confirmation that ExoX belongs to the DnaQ family. The active site of ExoX consists of four conserved acidic residues (Asp6 and Glu8 in β1, Asp85 in α4, Asp139 in α7) and His134 from the loop between α6 and α7, all of which participate in the cleavage process (Figure 1B).Figure 1.


Recognition and processing of double-stranded DNA by ExoX, a distributive 3'-5' exonuclease.

Wang T, Sun HL, Cheng F, Zhang XE, Bi L, Jiang T - Nucleic Acids Res. (2013)

Overall structure of complex I. (A) Ribbon diagram of ExoX in complex with 3′ overhanging dsDNA (ratio of 2:1). (B) Close-up view of the active site of ExoX; the DNA scissile strand twists and inserts into the active cavity of ExoX. Strong density peaks are observed at the two conserved metal-binding sites of the DnaQ family, suggesting the location of the two metal ions in ExoX. The difference (Fo-Fc) maps are contoured at 2.5σ.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3753628&req=5

gkt495-F1: Overall structure of complex I. (A) Ribbon diagram of ExoX in complex with 3′ overhanging dsDNA (ratio of 2:1). (B) Close-up view of the active site of ExoX; the DNA scissile strand twists and inserts into the active cavity of ExoX. Strong density peaks are observed at the two conserved metal-binding sites of the DnaQ family, suggesting the location of the two metal ions in ExoX. The difference (Fo-Fc) maps are contoured at 2.5σ.
Mentions: Although attempts to crystallize full-length ExoX failed, we were able to crystallize and solve the crystal structure of a C-terminal truncation of ExoX containing residues 1–167 (hereafter referred to as ExoX, Supplementary Figure S1), which retained activity. In each asymmetrical unit, two protein molecules (A and B) bind to the ends of a single dsDNA molecule, thus forming a dumbbell-like structure (Figure 1A). Each monomer of ExoX forms a bean-shaped core consisting of a central β-sheet (strands β1–β5) surrounded by eight α-helices (α1–α8), with helices α3–α4 on one side and helices α1–α2 and α5–α8 on the other side (Figure 1A). Analysis of the ExoX structure using the Dali server showed that ExoX has the highest degree of similarity to the ε subunit of DNA polymerase III (Z-score of 18.6, PDB code: 1J53). Despite these proteins sharing only ∼33% sequence identity, ExoX can be superimposed on the ε subunit of DNA polymerase III with a root-mean-square deviation for the Cα atoms of only 1.9 Å. This similarity, together with the presence of all three Exo motifs in ExoX (Supplementary Figure S2), provided further confirmation that ExoX belongs to the DnaQ family. The active site of ExoX consists of four conserved acidic residues (Asp6 and Glu8 in β1, Asp85 in α4, Asp139 in α7) and His134 from the loop between α6 and α7, all of which participate in the cleavage process (Figure 1B).Figure 1.

Bottom Line: When ExoX complexes with blunt-ended dsDNA or 5' overhanging dsDNA, a 'wedge' composed of Leu12 and Gln13 penetrates between the first two base pairs to break the 3' terminal base pair and facilitates precise feeding of the 3' terminus of the substrate strand into the ExoX cleavage active site.Site-directed mutagenesis showed that the complementary strand-binding site and the wedge of ExoX are dsDNA specific.Together with the results of structural comparisons, our data support a mechanism by which normal and mismatched dsDNA are recognized and digested by E. coli ExoX.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China, Graduate School of Chinese Academy of Sciences, Beijing 100039, China and State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.

ABSTRACT
Members of the DnaQ superfamily are major 3'-5' exonucleases that degrade either only single-stranded DNA (ssDNA) or both ssDNA and double-stranded DNA (dsDNA). However, the mechanism by which dsDNA is recognized and digested remains unclear. Exonuclease X (ExoX) is a distributive DnaQ exonuclease that cleaves both ssDNA and dsDNA substrates. Here, we report the crystal structures of Escherichia coli ExoX in complex with three different dsDNA substrates: 3' overhanging dsDNA, blunt-ended dsDNA and 3' recessed mismatch-containing dsDNA. In these structures, ExoX binds to dsDNA via both a conserved substrate strand-interacting site and a previously uncharacterized complementary strand-interacting motif. When ExoX complexes with blunt-ended dsDNA or 5' overhanging dsDNA, a 'wedge' composed of Leu12 and Gln13 penetrates between the first two base pairs to break the 3' terminal base pair and facilitates precise feeding of the 3' terminus of the substrate strand into the ExoX cleavage active site. Site-directed mutagenesis showed that the complementary strand-binding site and the wedge of ExoX are dsDNA specific. Together with the results of structural comparisons, our data support a mechanism by which normal and mismatched dsDNA are recognized and digested by E. coli ExoX. The crystal structures also provide insight into the structural framework of the different substrate specificities of the DnaQ family members.

Show MeSH
Related in: MedlinePlus