Structure of bacterial LigD 3′-phosphoesterase unveils a DNA repair superfamily

Abstract

The DNA ligase D (LigD) 3′-phosphoesterase (PE) module is a conserved component of the bacterial nonhomologous end-joining (NHEJ) apparatus that performs 3′ end-healing reactions at DNA double-strand breaks. Here we report the 1.9 Å crystal structure of Pseudomonas aeruginosa PE, which reveals that PE exemplifies a unique class of DNA repair enzyme. PE has a distinctive fold in which an eight stranded β barrel with a hydrophobic interior supports a crescent-shaped hydrophilic active site on its outer surface. Six essential side chains coordinate manganese and a sulfate mimetic of the scissile phosphate. The PE active site and mechanism are unique vis à vis other end-healing enzymes. We find PE homologs in archaeal and eukaryal proteomes, signifying that PEs comprise a DNA repair superfamily.is the key agent of the bacterial nonhomologous end-joining (NHEJ) pathway of DNA doublestrand break (DSB) repair (1). LigD is a single polypeptide consisting of three autonomous catalytic domain modules: an ATP-dependent ligase (LIG), a polymerase (POL), and a 3′-phosphoesterase (PE). The POL domain incorporates dNMP/rNMPs at DSB ends and gaps prior to strand sealing by the LIG domain (2-6) and is responsible, in large part, for the mutagenic outcomes of bacterial NHEJ in vivo (7). The PE domain provides a 3′ end-healing function, whereby it cleans up "dirty" DSBs with 3′-phosphate ends (8). PE also trims short 3′-ribonucleotide tracts (produced by POL) to generate the 3′ monoribonucleotide ends that are the preferred substrates for sealing by bacterial NHEJ ligases (3,8). The biochemical properties and atomic structures of the LIG and POL domains highlighted their membership in the covalent nucleotidyltransferase and archaeal/ eukaryal primase-polymerase families respectively (5, 9, 10). By contrast, the PE domain appears to be sui generis.The properties of the PE domain elucidated initially for Pseudomonas LigD also apply to the PE modules of Agrobacterium and Mycobacterium LigD (8,11,12). Specifically, PE displays a distinctive manganese-dependent 3′-ribonuclease/3′-phosphatase activity, entailing two component steps: (i) the 3′-terminal nucleoside is removed to yield a primer strand with a ribonucleoside 3′-PO 4 terminus; (ii) the 3′-PO 4 is hydrolyzed to a 3′-OH (Fig. 1A). PE activity is acutely dependent on the presence and length of a 5′ single-strand tail on a duplex primer-template substrate, thus implicating PE in 3′ end repair at gaps or recessed DSBs. Structure probing of Pseudomonas PE in solution revealed an apparently disordered N-terminal 29-aa segment, punctuated by a cluster of trypsin-and chymotrypsin-sensitive sites (Fig. 1B), flanking a seemingly well folded (i.e., protease insensitive) C-terminal domain (13). Deletion of the protease-sensitive N-terminal peptide had no effect on the phosphodiesterase activity of PE, though monoesterase activity was reduced. Mutational analyses identified an ensemble of conserved side chain functional groups within the protease-resistant module that were essential for ph...

DNA Ligase C1 Mediates the LigD-Independent Nonhomologous End-Joining Pathway of Mycobacterium smegmatis

“…To determine the role of the LigD-PE domain in the repair of chromosomal damage, we studied M. smegmatis carrying LigD with inactivating mutations in the LigD-PE domain, LigD-E310A and -H336A. The E310A substitution abolishes only phosphomonoesterase activity, whereas the H336A substitution abolishes both phosphomonoesterase and phosphodiesterase activities (19,23,24). Stationary-phase IR experiments indicated that PE inactivation through either mutation conferred a small but statistically significant sensitization to IR of approximately 5-fold at 1,200 Gy (Fig.…”

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

“…Atomic structures have been determined for the POL and LIG domains of bacterial LigD proteins bound to substrates at various functional states along their respective reaction pathways (Shuman and Glickman, 2007). On the other hand, only recently has the PE domain been characterized structurally (Nair et al, 2010). …”

“…We recently reported the 1.9 Å crystal structure of the active phosphodiesterase core of Pseudomonas aeruginosa PE ( Pae PE), spanning residues 30-187 (Nair et al, 2010). Pae PE has a novel fold in which an 8 stranded β-barrel with a hydrophobic interior supports a crescent-shaped hydrophilic active site on its outer surface.…”

Ribonucleolytic resection is required for repair of strand displaced nonhomologous end-joining intermediates

“…Many archaeal species survive in some of the most extreme environments on the planet, and therefore it is important to investigate how they maintain genome stability to understand how life evolved in the hostile conditions present on the primordial earth and how these organisms continue to thrive in extreme terrestrial niches. Although some NHEJ-like genes have been identified in archaea (9,(16)(17)(18), a complete DNA-break repair apparatus has not been reported. Therefore, it is unclear if a related DSBrepair pathway operates in archaea.…”