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

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•Mn2+• 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 (Cd2+) and inhibitory (Zn2+) metals provide evidence for significant conformational changes at and near the active site, compared to the relatively modest changes elicited by Mn2+.

Molecular basis for DNA strand displacement by NHEJ repair polymerases

“…The architecture of the structures implies that the surface loop is flexible and may enclose the DNA once the proper substrate is engaged for catalysis (Supplementary Figure S1E,1F). This speculation is supported by NMR solution studies that suggest that the crystal structures represents a ‘closed’ active site formation, and that the loop moves away from the active site in the absence of a substrate ( 33 ). This study also showed that upon addition of substrate, spectral perturbations were observed that indicated movement of the loop region, and formation of a ‘closed’ active site from an ‘open’ one.…”

Substrate-Dependent Millisecond Domain Motions in DNA Polymerase β

“…32 More recently, backbone flexibility in DNA ligase D was suggested to be involved in both activation and inhibition of this enzyme. 33 …”

Structural Model of the Extracellular Assembly of the TCR-CD3 Complex

“…In each titration case, assignments were carefully transferred from the CD3 free TROSY spectra to saturated TROSY spectra. The scaled chemical shift, δppm = ((Δ 1 H) 2 +0.11(Δ 15 N) 2 ) 0.5 (Natarajan et al, 2012). The residues that showed spectral changes were identified if (a) they showed scaled chemical shift changes greater than 1σ above the 25% trimmed mean deviation when comparing in the presence and absence of saturated amounts of CD3 state, and (b) lesser than 1σ below the 25% trimmed mean deviation in the ratio of NMR signal intensity between CD3 saturated-state and CD3 free state (Figure 2).…”