The gene symbol ligD is unambiguous and correctly matches the UniProt description for the target.
All identifiers, the enzyme class, the domain complement, and the organism are internally consistent and match the well-characterized bacterial NHEJ DNA ligase D family. A study performed directly in P. putida (Paris et al., 2015, PMID 25942369) explicitly describes the LigD/Ku NHEJ system in this organism, and the cognate Ku gene is present in the same genome (PP_3255; see below). There is no evidence of gene-symbol ambiguity — this is the canonical bacterial NHEJ multifunctional ligase, not an unrelated same-symbol gene.
LigD is the central, multifunctional, ATP-dependent DNA ligase of the bacterial non-homologous end-joining (NHEJ) pathway. Its primary function is to repair chromosomal DNA double-strand breaks (DSBs) without a homologous template, acting as the enzymatic "workhorse" downstream of the DNA-end-binding protein Ku. LigD is a single polypeptide carrying three autonomous catalytic modules that together perform every chemical step of end-joining: a 3'-phosphoesterase (PE) domain that heals damaged 3' ends to a ligatable 3'-OH; an ATP-dependent ligase (LIG) domain that seals the phosphodiester backbone via a covalent ligase–AMP intermediate; and a primase/polymerase (POL) domain that fills gaps and adds (ribo)nucleotides across the break. The enzyme functions in the cytoplasm on nucleoid (chromosomal) DNA, and is physiologically most important in non-replicating / stationary-phase / starved cells, where homologous recombination is unavailable. NHEJ by Ku–LigD is intrinsically error-prone, so LigD also contributes to stress-associated (stationary-phase) mutagenesis in P. putida.
Bacterial NHEJ is a minimal two-component system: the homodimeric end-binding protein Ku plus the polyfunctional ligase LigD possess all the break-recognition, end-processing and ligation activities needed to rejoin a DSB (Gong et al., 2005, PMID 15778718; Pitcher, Brissett & Doherty, 2007, PMID 17506672; Amare et al., 2021, PMID 34901162). Ku binds and synapses the two broken ends and recruits/stimulates LigD, which then processes and seals the junction (Amare et al., 2021, PMID 34901162; Zhu & Shuman, 2010, PMID 20018881).
InterPro/Pfam mapping of the 833-aa Q88HU3 sequence gives an unambiguous N→C module order, matching the experimentally characterized Pseudomonas aeruginosa LigD:
| Module | Approx. residues | InterPro / Pfam | Catalytic role |
|---|---|---|---|
| PE (3'-phosphoesterase) | ~5–160 | IPR014144 / PF13298 | End-healing: generate 3'-OH |
| LIG (ATP-dependent ligase) | ~219–520 (central IPR012310/PF01068 + OB-fold C-term IPR012309/PF04679) | IPR014146 | Nick sealing |
| POL (primase-polymerase) | ~547–812 (PaeLigD-type IPR033651) | IPR014145 / PF21686 | Gap fill / nucleotide addition |
The POL module is explicitly of the PaeLigD-type subclass, i.e., the same structural class as the crystallized P. aeruginosa POL domain, allowing high-confidence transfer of the P. aeruginosa biochemistry to the P. putida ortholog (Iteration 2 analysis).
The N-terminal PE module catalyzes Mn-dependent 3'-phosphodiesterase and 3'-phosphomonoesterase reactions at the primer-strand 3' end (Zhu & Shuman, 2006, PMID 16540477). It acts as a 3'-exoribonuclease that resects a 3'-terminal diribonucleotide to a ribonucleoside-3'-PO₄ (strictly requiring the 2'-OH of the penultimate ribose), and its phosphomonoesterase then converts a 3'-PO₄ (ribo- or deoxyribo-) to a ligatable 3'-OH (Zhu & Shuman, 2006, PMID 16540477). This "heals" damaged ends and removes the ribonucleotides deposited by POL, coordinating the processing→sealing hand-off.
The ligase module is an ATP-dependent DNA ligase that performs the final sealing step. The structure of the LigD ligase domain was captured as the covalent ligase–AMP (adenylylated-enzyme) intermediate with a divalent metal in the active site (Akey et al., 2006, PMID 16476729), consistent with canonical two-step ligase chemistry: (i) enzyme adenylylation using ATP, (ii) AMP transfer to the DNA 5'-phosphate, (iii) phosphodiester bond formation joining 3'-OH and 5'-PO₄. Its activity is dynamically balanced against end-remodeling, which underlies NHEJ's error-prone signature (Akey et al., 2006, PMID 16476729).
Net reaction (EC 6.5.1.1): ATP + (5'-phospho-DNA)ₙ + (3'-hydroxy-DNA)ₘ → AMP + diphosphate + ligated DNA — carried out at DSB junctions after PE/POL end-processing.
LigD is a soluble cytoplasmic enzyme that acts on chromosomal (nucleoid) DNA. There is no signal peptide, transmembrane region, or secretion signal; the protein's substrate is intracellular double-stranded DNA. Functionally it is recruited to double-strand breaks in the bacterial chromosome, where Ku first binds the DNA ends and delivers LigD (Amare et al., 2021, PMID 34901162; Zhu & Shuman, 2010, PMID 20018881). Genetic work in P. putida demonstrates its action on the resident chromosome under starvation (Paris et al., 2015, PMID 25942369).
Supported:
1. LigD is the multifunctional ATP-dependent DNA ligase of bacterial NHEJ (EC 6.5.1.1). ✔
2. It contains three separable catalytic modules (PE, LIG, POL) in N→C order PE–LIG–POL. ✔ (bioinformatically confirmed for Q88HU3)
3. POL is an AEP/primase-fold, Mn-dependent, ribonucleotide-preferring gap-filling polymerase that senses 5'-phosphate. ✔
4. PE is a 3'-phosphoesterase/exoribonuclease generating 3'-OH ends. ✔
5. It acts in the cytoplasm on chromosomal DNA, primarily in non-replicating/starved cells, and contributes to error-prone stationary-phase mutagenesis in P. putida. ✔
6. A cognate Ku partner is genomically encoded (PP_3255) — complete two-component system. ✔
Refuted / not supported:
- The gene-symbol-ambiguity concern is refuted — ligD unambiguously denotes the NHEJ ligase for this accession/organism.
Report generated across Iterations 1–3. Citations reference PubMed IDs of the primary literature and reviews surveyed.