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Solution structure and DNA-binding properties of the phosphoesterase domain of DNA ligase D.

Natarajan A, Dutta K, Temel DB, Nair PA, Shuman S, Ghose R - Nucleic Acids Res. (2011)

Bottom Line: PE exemplifies a new family of DNA end-healing enzymes found in all phylogenetic domains.PaePE has a disordered N-terminus and a well-folded core that differs in instructive ways from the crystal structure of a PaePE•Mn(2+)• sulfate complex, especially at the active site that is found to be conformationally dynamic.Spectral perturbations measured in the presence of weakly catalytic (Cd(2+)) and inhibitory (Zn(2+)) metals provide evidence for significant conformational changes at and near the active site, compared to the relatively modest changes elicited by Mn(2+).

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, The City College of New York, 160 Convent Avenue, New York, NY 10031, USA.

ABSTRACT
The phosphoesterase (PE) domain of the bacterial DNA repair enzyme LigD possesses distinctive manganese-dependent 3'-phosphomonoesterase and 3'-phosphodiesterase activities. PE exemplifies a new family of DNA end-healing enzymes found in all phylogenetic domains. Here, we determined the structure of the PE domain of Pseudomonas aeruginosa LigD (PaePE) using solution NMR methodology. PaePE has a disordered N-terminus and a well-folded core that differs in instructive ways from the crystal structure of a PaePE•Mn(2+)• sulfate complex, especially at the active site that is found to be conformationally dynamic. Chemical shift perturbations in the presence of primer-template duplexes with 3'-deoxynucleotide, 3'-deoxynucleotide 3'-phosphate, or 3' ribonucleotide termini reveal the surface used by PaePE to bind substrate DNA and suggest a more efficient engagement in the presence of a 3'-ribonucleotide. Spectral perturbations measured in the presence of weakly catalytic (Cd(2+)) and inhibitory (Zn(2+)) metals provide evidence for significant conformational changes at and near the active site, compared to the relatively modest changes elicited by Mn(2+).

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15N-{1H} Steady-state NOE for PaePE. Data was acquired at 600 MHz. NOE values for the β-strands and the α-helix are shown in red and blue respectively.
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gkr950-F1: 15N-{1H} Steady-state NOE for PaePE. Data was acquired at 600 MHz. NOE values for the β-strands and the α-helix are shown in red and blue respectively.

Mentions: Inspection of the final NMR ensemble of 15 structures (see Table 1 for statistics) reveals a disordered N-terminus (1–31). This fact is also borne out by the extremely low 15N-{1H} NOE values (0.08 ± 0.21; see Figure 1) for this region. However, some degree of local ordering is seen encompassing residues Asp15–Thr19 (15N-{1H} NOE: 0.18 ± 0.01). This region contains Asp15 and is flanked by Arg14 and Glu21; these three sidechains are important for the 3′-monoesterase activity of PaePE (16). The distribution of ϕ/ψ angles for the Asp15–Thr19 segment indicates an overall extended structure (Supplementary Figure S1). A hydrogen bond between the carbonyl oxygen of Thr19 and the amide of Glu21 is found in all 15 structures of the final ensemble.Figure 1.


Solution structure and DNA-binding properties of the phosphoesterase domain of DNA ligase D.

Natarajan A, Dutta K, Temel DB, Nair PA, Shuman S, Ghose R - Nucleic Acids Res. (2011)

15N-{1H} Steady-state NOE for PaePE. Data was acquired at 600 MHz. NOE values for the β-strands and the α-helix are shown in red and blue respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3300020&req=5

gkr950-F1: 15N-{1H} Steady-state NOE for PaePE. Data was acquired at 600 MHz. NOE values for the β-strands and the α-helix are shown in red and blue respectively.
Mentions: Inspection of the final NMR ensemble of 15 structures (see Table 1 for statistics) reveals a disordered N-terminus (1–31). This fact is also borne out by the extremely low 15N-{1H} NOE values (0.08 ± 0.21; see Figure 1) for this region. However, some degree of local ordering is seen encompassing residues Asp15–Thr19 (15N-{1H} NOE: 0.18 ± 0.01). This region contains Asp15 and is flanked by Arg14 and Glu21; these three sidechains are important for the 3′-monoesterase activity of PaePE (16). The distribution of ϕ/ψ angles for the Asp15–Thr19 segment indicates an overall extended structure (Supplementary Figure S1). A hydrogen bond between the carbonyl oxygen of Thr19 and the amide of Glu21 is found in all 15 structures of the final ensemble.Figure 1.

Bottom Line: PE exemplifies a new family of DNA end-healing enzymes found in all phylogenetic domains.PaePE has a disordered N-terminus and a well-folded core that differs in instructive ways from the crystal structure of a PaePE•Mn(2+)• sulfate complex, especially at the active site that is found to be conformationally dynamic.Spectral perturbations measured in the presence of weakly catalytic (Cd(2+)) and inhibitory (Zn(2+)) metals provide evidence for significant conformational changes at and near the active site, compared to the relatively modest changes elicited by Mn(2+).

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, The City College of New York, 160 Convent Avenue, New York, NY 10031, USA.

ABSTRACT
The phosphoesterase (PE) domain of the bacterial DNA repair enzyme LigD possesses distinctive manganese-dependent 3'-phosphomonoesterase and 3'-phosphodiesterase activities. PE exemplifies a new family of DNA end-healing enzymes found in all phylogenetic domains. Here, we determined the structure of the PE domain of Pseudomonas aeruginosa LigD (PaePE) using solution NMR methodology. PaePE has a disordered N-terminus and a well-folded core that differs in instructive ways from the crystal structure of a PaePE•Mn(2+)• sulfate complex, especially at the active site that is found to be conformationally dynamic. Chemical shift perturbations in the presence of primer-template duplexes with 3'-deoxynucleotide, 3'-deoxynucleotide 3'-phosphate, or 3' ribonucleotide termini reveal the surface used by PaePE to bind substrate DNA and suggest a more efficient engagement in the presence of a 3'-ribonucleotide. Spectral perturbations measured in the presence of weakly catalytic (Cd(2+)) and inhibitory (Zn(2+)) metals provide evidence for significant conformational changes at and near the active site, compared to the relatively modest changes elicited by Mn(2+).

Show MeSH