Sequence-specific 1H, 13C and 15N assignments of the phosphoesterase (PE) domain of Pseudomonas aeruginosa DNA ligase D (LigD)

Abstract

DNA ligase D (LigD), consisting of polymerase, ligase and phosphoesterase domains, is the essential catalyst of the bacterial non-homologous end-joining pathway of DNA double-strand break repair. The phosphoesterase (PE) module performs manganese-dependent 3’-phosphomonoesterase and 3’-ribonucleoside resection reactions that heal broken ends in preparation for sealing. LigD PE exemplifies a structurally and mechanistically unique class of DNA end-processing enzymes. Here, we present the resonance assignments of the PE domain of Pseudomonas aeruginosa LigD comprising the N-terminal 177 residues.

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

“…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 15 N-{ 1 H} NOE values (0.08 ± 0.21; see Figure 1 ) for this region.…”

“…Pae PE-(1–177) (referred to hereafter as Pae PE) was produced in E. coli as an N-terminal His 10 -Smt3 fusion; isotope labeling and purification of the tag-free Pae PE protein are described in detail elsewhere ( 19 ). Pae PE samples for NMR experiments were prepared in a buffer containing 50 mM Bis–Tris (pH 6.5), 200 mM NaCl and 5 mM DTT (referred to as the NMR buffer from hereon forward) and typically contained ~300 µM protein.…”

“…Sidechain 13 C and 1 H assignments were obtained via (H)C(CO)NH and H(CCO)NH experiments utilizing uniformly 13 C, 15 N-labeled Pae PE ( 21 ). Experimental details have been described at length elsewhere ( 19 ).…”

“…Initial NMR studies were performed with Pae PE-(1–187), which produced spectra that were suitable for obtaining backbone assignments, but were not of high enough quality to yield a sufficient number of sidechain 1 H assignments to allow structure determination. Serial C-terminal truncations identified Pae PE-(1–177) as a catalytically active 3′-monoesterase/3′-diesterase that produced spectra of considerably higher quality and yielded sufficient number of 1 H assignments ( 19 ) suitable for calculating an NMR structure of this highly dynamic enzymatic module. Here, we present the solution structure of Pae PE-(1–177) determined using standard NOE-based approaches.…”

Structural insights to the metal specificity of an archaeal member of the LigD 3′-phosphoesterase DNA repair enzyme family

“…Missing from the Pae PE crystal structure was an N-terminal peptide segment, deletion of which selectively diminished the 3′-phosphomonoesterase activity of Pae PE without affecting the 3′-phosphodiesterase ( 2 ). Protease-sensitivity and solution NMR studies of bacterial PE show that the N-terminal peptide is disordered ( 2 , 14 ).…”