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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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Protein–DNA interactions in complexes I and II. Schematic diagram of the protein–DNA interactions in complexes I (A) and II (C). (B) Close-up view of the five interactions between ExoX and the 3′ overhanging dsDNA. (D) Close-up view of the three interactions between ExoX and the blunt duplex DNA complementary strand. Interactions exist between Arg87 and the complementary DNA strand in complex II that cannot be observed in complex I.
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gkt495-F2: Protein–DNA interactions in complexes I and II. Schematic diagram of the protein–DNA interactions in complexes I (A) and II (C). (B) Close-up view of the five interactions between ExoX and the 3′ overhanging dsDNA. (D) Close-up view of the three interactions between ExoX and the blunt duplex DNA complementary strand. Interactions exist between Arg87 and the complementary DNA strand in complex II that cannot be observed in complex I.

Mentions: In complex I, the 3′ overhang is bent at an angle of 70 degrees between the first two nucleotides and extends into the active site (Figure 1B). Outside the active site, a substrate DNA strand-binding site, mainly composed of Lys111, Tyr112 and Asn114 in the α5–α6 loop, interacts with the sugar–phosphate backbone of the substrate strand (Figures 2A and B). This site is similar to the ssDNA binding sites observed in other DnaQ family members (16). Moreover, Lys101 and Arg104 of α5 form a specific positively charged binding site for the complementary strand. Most of the residues formed electrostatic interactions or hydrogen bonds with the DNA sugar–phosphate backbone, while few displayed electrostatic interactions or hydrogen bonding with DNA bases, which is indicative of a non–sequence-specific DNA-binding site (Figures 2A and B). Strong density peaks were observed at the two conserved metal-binding sites of the DnaQ family, A and B (8,25) (Figure 1B). The intact substrate observed in our structure suggest that the metals involved are unlikely to be active metals (such as Mn2+ or Mg2+), and are probably inactive metals (such as Ni2+ or Na+). Although we cannot be certain of the exact metal ion species present, the undefined metal ion is of no consequence for the following discussion.Figure 2.


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)

Protein–DNA interactions in complexes I and II. Schematic diagram of the protein–DNA interactions in complexes I (A) and II (C). (B) Close-up view of the five interactions between ExoX and the 3′ overhanging dsDNA. (D) Close-up view of the three interactions between ExoX and the blunt duplex DNA complementary strand. Interactions exist between Arg87 and the complementary DNA strand in complex II that cannot be observed in complex I.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt495-F2: Protein–DNA interactions in complexes I and II. Schematic diagram of the protein–DNA interactions in complexes I (A) and II (C). (B) Close-up view of the five interactions between ExoX and the 3′ overhanging dsDNA. (D) Close-up view of the three interactions between ExoX and the blunt duplex DNA complementary strand. Interactions exist between Arg87 and the complementary DNA strand in complex II that cannot be observed in complex I.
Mentions: In complex I, the 3′ overhang is bent at an angle of 70 degrees between the first two nucleotides and extends into the active site (Figure 1B). Outside the active site, a substrate DNA strand-binding site, mainly composed of Lys111, Tyr112 and Asn114 in the α5–α6 loop, interacts with the sugar–phosphate backbone of the substrate strand (Figures 2A and B). This site is similar to the ssDNA binding sites observed in other DnaQ family members (16). Moreover, Lys101 and Arg104 of α5 form a specific positively charged binding site for the complementary strand. Most of the residues formed electrostatic interactions or hydrogen bonds with the DNA sugar–phosphate backbone, while few displayed electrostatic interactions or hydrogen bonding with DNA bases, which is indicative of a non–sequence-specific DNA-binding site (Figures 2A and B). Strong density peaks were observed at the two conserved metal-binding sites of the DnaQ family, A and B (8,25) (Figure 1B). The intact substrate observed in our structure suggest that the metals involved are unlikely to be active metals (such as Mn2+ or Mg2+), and are probably inactive metals (such as Ni2+ or Na+). Although we cannot be certain of the exact metal ion species present, the undefined metal ion is of no consequence for the following discussion.Figure 2.

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