1H, 15N and 13C resonance backbone and side-chain assignments and secondary structure determination of the BRCT domain of Mtb LigA.

The BRCA1 carboxyl-terminal (BRCT) domain, an evolutionarily conserved structural motif, is ubiquitous in a multitude of proteins spanning prokaryotic and eukaryotic organisms. In Mycobacterium tuberculosis (Mtb), BRCT domain plays a pivotal role in the catalytic activity of the NAD+-dependent DNA ligase (LigA). LigA is pivotal in DNA replication, catalyzing the formation of phosphodiester bonds in Okazaki fragments and repairing single-strand breaks in damaged DNA, essential for the survival of Mtb. Structural and functional aspects of LigA unveil its character as a highly modular protein, undergoing substantial conformational changes during its catalytic cycle. Although the BRCT domain of Mtb LigA plays an essential role in DNA binding and protein-protein interactions, the precise mechanism of action remains poorly understood. Unravelling the structure of the BRCT domain holds the promise of advancing our understanding of this pivotal domain. Additionally, it will facilitate further exploration of the protein-protein interactions and enhance our understanding of inter domain interactions within LigA, specifically between BRCT and the Adenylation domain. In this study, we demonstrate the overexpression of the BRCT domain of Mtb LigA and conduct its analysis using solution NMR spectroscopy, revealing a well-folded structure and we present the nearly complete chemical shift assignments of both backbone and sidechains. In addition, a secondary structure prediction by TALOS N predicts BRCT consisting of 3 α-helices and 4 ß-sheets, closely resembling the typical structural topology of most BRCT domains.

FUS unveiled in mitochondrial DNA repair and targeted ligase-1 expression rescues repair-defects in FUS-linked motor neuron disease.

This study establishes the physiological role of Fused in Sarcoma (FUS) in mitochondrial DNA (mtDNA) repair and highlights its implications to the pathogenesis of FUS-associated neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Endogenous FUS interacts with and recruits mtDNA Ligase IIIα (mtLig3) to DNA damage sites within mitochondria, a relationship essential for maintaining mtDNA repair and integrity in healthy cells. Using ALS patient-derived FUS mutant cell lines, a transgenic mouse model, and human autopsy samples, we discovered that compromised FUS functionality hinders mtLig3's repair role, resulting in increased mtDNA damage and mutations. These alterations cause various manifestations of mitochondrial dysfunction, particularly under stress conditions relevant to disease pathology. Importantly, rectifying FUS mutations in patient-derived induced pluripotent cells (iPSCs) preserves mtDNA integrity. Similarly, targeted introduction of human DNA Ligase 1 restores repair mechanisms and mitochondrial activity in FUS mutant cells, suggesting a potential therapeutic approach. Our findings unveil FUS's critical role in mitochondrial health and mtDNA repair, offering valuable insights into the mechanisms underlying mitochondrial dysfunction in FUS-associated motor neuron disease.

DNA-PKcs suppresses illegitimate chromosome rearrangements.

Two DNA repair pathways, non-homologous end joining (NHEJ) and alternative end joining (A-EJ), are involved in V(D)J recombination and chromosome translocation. Previous studies reported distinct repair mechanisms for chromosome translocation, with NHEJ involved in humans and A-EJ in mice predominantly. NHEJ depends on DNA-PKcs, a critical partner in synapsis formation and downstream component activation. While DNA-PKcs inhibition promotes chromosome translocations harboring microhomologies in mice, its synonymous effect in humans is not known. We find partial DNA-PKcs inhibition in human cells leads to increased translocations and the continued involvement of a dampened NHEJ. In contrast, complete DNA-PKcs inhibition substantially increased microhomology-mediated end joining (MMEJ), thus bridging the two different translocation mechanisms between human and mice. Similar to a previous study on Ku70 deletion, DNA-PKcs deletion in G1/G0-phase mouse progenitor B cell lines, significantly impairs V(D)J recombination and generated higher rates of translocations as a consequence of dysregulated coding and signal end joining. Genetic DNA-PKcs inhibition suppresses NHEJ entirely, with repair phenotypically resembling Ku70-deficient A-EJ. In contrast, we find DNA-PKcs necessary in generating the near-exclusive MMEJ associated with Lig4 deficiency. Our study underscores DNA-PKcs in suppressing illegitimate chromosome rearrangement while also contributing to MMEJ in both species.

Unfilled gaps by polß lead to aberrant ligation by LIG1 at the downstream steps of base excision repair pathway.

Base excision repair (BER) involves the tightly coordinated function of DNA polymerase ß (polß) and DNA ligase I (LIG1) at the downstream steps. Our previous studies emphasize that defective substrate-product channeling, from gap filling by polß to nick sealing by LIG1, can lead to interruptions in repair pathway coordination. Yet, the molecular determinants that dictate accurate BER remains largely unknown. Here, we demonstrate that a lack of gap filling by polß leads to faulty repair events and the formation of deleterious DNA intermediates. We dissect how ribonucleotide challenge and cancer-associated mutations could adversely impact the ability of polß to efficiently fill the one nucleotide gap repair intermediate which subsequently results in gap ligation by LIG1, leading to the formation of single-nucleotide deletion products. Moreover, we demonstrate that LIG1 is not capable of discriminating against nick DNA containing a 3'-ribonucleotide, regardless of base-pairing potential or damage. Finally, AP-Endonuclease 1 (APE1) shows distinct substrate specificity for the exonuclease removal of 3'-mismatched bases and ribonucleotides from nick repair intermediate. Overall, our results reveal that unfilled gaps result in impaired coordination between polß and LIG1, defining a possible type of mutagenic event at the downstream steps where APE1 could provide a proofreading role to maintain BER efficiency.

Structural role for DNA Ligase IV in promoting the fidelity of non-homologous end joining.

Nonhomologous end joining (NHEJ), the primary pathway of vertebrate DNA double-strand-break (DSB) repair, directly re-ligates broken DNA ends. Damaged DSB ends that cannot be immediately re-ligated are modified by NHEJ processing enzymes, including error-prone polymerases and nucleases, to enable ligation. However, DSB ends that are initially compatible for re-ligation are typically joined without end processing. As both ligation and end processing occur in the short-range (SR) synaptic complex that closely aligns DNA ends, it remains unclear how ligation of compatible ends is prioritized over end processing. In this study, we identify structural interactions of the NHEJ-specific DNA Ligase IV (Lig4) within the SR complex that prioritize ligation and promote NHEJ fidelity. Mutational analysis demonstrates that Lig4 must bind DNA ends to form the SR complex. Furthermore, single-molecule experiments show that a single Lig4 binds both DNA ends at the instant of SR synapsis. Thus, Lig4 is poised to ligate compatible ends upon initial formation of the SR complex before error-prone processing. Our results provide a molecular basis for the fidelity of NHEJ.

Horizontal acquisition of a DNA ligase improves DNA damage tolerance in eukaryotes.

Bdelloid rotifers are part of the restricted circle of multicellular animals that can withstand a wide range of genotoxic stresses at any stage of their life cycle. In this study, bdelloid rotifer Adineta vaga is used as a model to decipher the molecular basis of their extreme tolerance. Proteomic analysis shows that a specific DNA ligase, different from those usually involved in DNA repair in eukaryotes, is strongly over-represented upon ionizing radiation. A phylogenetic analysis reveals its orthology to prokaryotic DNA ligase E, and its horizontal acquisition by bdelloid rotifers and plausibly other eukaryotes. The fungus Mortierella verticillata, having a single copy of this DNA Ligase E homolog, also exhibits an increased radiation tolerance with an over-expression of this DNA ligase E following X-ray exposure. We also provide evidence that A. vaga ligase E is a major contributor of DNA breaks ligation activity, which is a common step of all important DNA repair pathways. Consistently, its heterologous expression in human cell lines significantly improves their radio-tolerance. Overall, this study highlights the potential of horizontal gene transfers in eukaryotes, and their contribution to the adaptation to extreme conditions.

Deficiency of ligase IV leads to reduced NHEJ, accumulation of DNA damage, and can sensitize cells to cancer therapeutics.

Ligase IV is a key enzyme involved during DNA double-strand breaks (DSBs) repair through nonhomologous end joining (NHEJ). However, in contrast to Ligase IV deficient mouse cells, which are embryonic lethal, Ligase IV deficient human cells, including pre-B cells, are viable. Using CRISPR-Cas9 mediated genome editing, we have generated six different LIG4 mutants in cervical cancer and normal kidney epithelial cell lines. While the LIG4 mutant cells showed a significant reduction in NHEJ, joining mediated through microhomology-mediated end joining (MMEJ) and homologous recombination (HR) were significantly high. The reduced NHEJ joining activity was restored by adding purified Ligase IV/XRCC4. Accumulation of DSBs and reduced cell viability were observed in LIG4 mutant cells. LIG4 mutant cells exhibited enhanced sensitivity towards DSB-inducing agents such as ionizing radiation (IR) and etoposide. More importantly, the LIG4 mutant of cervical cancer cells showed increased sensitivity towards FDA approved drugs such as Carboplatin, Cisplatin, Paclitaxel, Doxorubicin, and Bleomycin used for cervical cancer treatment. These drugs, in combination with IR showed enhanced cancer cell death in the background of LIG4 gene mutation. Thus, our study reveals that mutation in LIG4 results in compromised NHEJ, leading to sensitization of cervical cancer cells towards currently used cancer therapeutics.

Molecular cloning and characterization of Pcal_0039, an ATP-/NAD+-independent DNA ligase from hyperthermophilic archaeon Pyrobaculum calidifontis.

The genome sequence of hyperthermophilic archaeon Pyrobaculum calidifontis contains an open reading frame, Pcal_0039, which encodes a putative DNA ligase. Structural analysis disclosed the presence of signature sequences of ATP-dependent DNA ligases. We have heterologously expressed Pcal_0039 gene in Escherichia coli. The recombinant protein, majorly produced in soluble form, was purified and functionally characterized. Recombinant Pcal_0039 displayed nick-joining activity between 40 and 85 °C. Optimal activity was observed at 70 °C and pH 5.5. Nick-joining activity was retained even after heating for 1 h at 90 °C, indicating highly thermostable nature of Pcal_0039. The nick-joining activity, displayed by Pcal_0039, was metal ion dependent and Mg2+ was the most preferred. NaCl and KCl inhibited the nick-joining activity at or above 200 mmol/L. The activity catalyzed by recombinant Pcal_0039 was independent of addition of ATP or NAD+ or any other nucleotide cofactor. A mismatch adjacent to the nick, either at 3'- or 5'-end, abolished the nick-joining activity. These characteristics make Pcal_0039 a potential candidate for applications in DNA diagnostics. To the best of our knowledge, Pcal_0039 is the only DNA ligase, characterized from genus Pyrobaculum, which exhibits optimum nick-joining activity at pH below 6.0 and independent of any nucleotide cofactor.

A four-point molecular handover during Okazaki maturation.

DNA replication introduces thousands of RNA primers into the lagging strand that need to be removed for replication to be completed. In Escherichia coli when the replicative DNA polymerase Pol IIIα terminates at a previously synthesized RNA primer, DNA Pol I takes over and continues DNA synthesis while displacing the downstream RNA primer. The displaced primer is subsequently excised by an endonuclease, followed by the sealing of the nick by a DNA ligase. Yet how the sequential actions of Pol IIIα, Pol I polymerase, Pol I endonuclease and DNA ligase are coordinated is poorly defined. Here we show that each enzymatic activity prepares the DNA substrate for the next activity, creating an efficient four-point molecular handover. The cryogenic-electron microscopy structure of Pol I bound to a DNA substrate with both an upstream and downstream primer reveals how it displaces the primer in a manner analogous to the monomeric helicases. Moreover, we find that in addition to its flap-directed nuclease activity, the endonuclease domain of Pol I also specifically cuts at the RNA-DNA junction, thus marking the end of the RNA primer and creating a 5' end that is a suitable substrate for the ligase activity of LigA once all RNA has been removed.

Plant PAXX has an XLF-like function and stimulates DNA end joining by the Ku-DNA ligase IV/XRCC4 complex.

Non-homologous end joining (NHEJ) plays a major role in repairing DNA double-strand breaks and is key to genome stability and editing. The minimal core NHEJ proteins, namely Ku70, Ku80, DNA ligase IV and XRCC4, are conserved, but other factors vary in different eukaryote groups. In plants, the only known NHEJ proteins are the core factors, while the molecular mechanism of plant NHEJ remains unclear. Here, we report a previously unidentified plant ortholog of PAXX, the crystal structure of which showed a similar fold to human 'PAXX'. However, plant PAXX has similar molecular functions to human XLF, by directly interacting with Ku70/80 and XRCC4. This suggests that plant PAXX combines the roles of mammalian PAXX and XLF and that these functions merged into a single protein during evolution. This is consistent with a redundant function of PAXX and XLF in mammals.

Unchanged PCNA and DNMT1 dynamics during replication in DNA ligase I-deficient cells but abnormal chromatin levels of non-replicative histone H1.

DNA ligase I (LigI), the predominant enzyme that joins Okazaki fragments, interacts with PCNA and Pol δ. LigI also interacts with UHRF1, linking Okazaki fragment joining with DNA maintenance methylation. Okazaki fragments can also be joined by a relatively poorly characterized DNA ligase IIIα (LigIIIα)-dependent backup pathway. Here we examined the effect of LigI-deficiency on proteins at the replication fork. Notably, LigI-deficiency did not alter the kinetics of association of the PCNA clamp, the leading strand polymerase Pol ε, DNA maintenance methylation proteins and core histones with newly synthesized DNA. While the absence of major changes in replication and methylation proteins is consistent with the similar proliferation rate and DNA methylation levels of the LIG1 null cells compared with the parental cells, the increased levels of LigIIIα/XRCC1 and Pol δ at the replication fork and in bulk chromatin indicate that there are subtle replication defects in the absence of LigI. Interestingly, the non-replicative histone H1 variant, H1.0, is enriched in the chromatin of LigI-deficient mouse CH12F3 and human 46BR.1G1 cells. This alteration was not corrected by expression of wild type LigI, suggesting that it is a relatively stable epigenetic change that may contribute to the immunodeficiencies linked with inherited LigI-deficiency syndrome.

The DNA binding domain and the C-terminal region of DNA Ligase IV specify its role in V(D)J recombination.

DNA Ligase IV is responsible for the repair of DNA double-strand breaks (DSB), including DSBs that are generated during V(D)J recombination. Like other DNA ligases, Ligase IV contains a catalytic core with three subdomains-the DNA binding (DBD), the nucleotidyltransferase (NTD), and the oligonucleotide/oligosaccharide-fold subdomain (OBD). Ligase IV also has a unique C-terminal region that includes two BRCT domains, a nuclear localization signal sequence and a stretch of amino acid that participate in its interaction with XRCC4. Out of the three mammalian ligases, Ligase IV is the only ligase that participates in and is required for V(D)J recombination. Identification of the minimal domains within DNA Ligase IV that contribute to V(D)J recombination has remained unresolved. The interaction of the Ligase IV DNA binding domain with Artemis, and the interaction of its C-terminal region with XRCC4, suggest that both of these regions that also interact with the Ku70/80 heterodimer are important and might be sufficient for mediating participation of DNA Ligase IV in V(D)J recombination. This hypothesis was investigated by generating chimeric ligase proteins by swapping domains, and testing their ability to rescue V(D)J recombination in Ligase IV-deficient cells. We demonstrate that a fusion protein containing Ligase I NTD and OBDs flanked by DNA Ligase IV DBD and C-terminal region is sufficient to support V(D)J recombination. This chimeric protein, which we named Ligase 37, complemented formation of coding and signal joints. Coding joints generated with Ligase 37 were shorter than those observed with wild type DNA Ligase IV. The shorter length was due to increased nucleotide deletions and decreased nucleotide insertions. Additionally, overexpression of Ligase 37 in a mouse pro-B cell line supported a shift towards shorter coding joints. Our findings demonstrate that the ability of DNA Ligase IV to participate in V(D)J recombination is in large part mediated by its DBD and C-terminal region.

Human DNA ligases I and III have stand-alone end-joining capability, but differ in ligation efficiency and specificity.

Double-strand DNA breaks (DSBs) are toxic to cells, and improper repair can cause chromosomal abnormalities that initiate and drive cancer progression. DNA ligases III and IV (LIG3, LIG4) have long been credited for repair of DSBs in mammals, but recent evidence suggests that DNA ligase I (LIG1) has intrinsic end-joining (EJ) activity that can compensate for their loss. To test this model, we employed in vitro biochemical assays to compare EJ by LIG1 and LIG3. The ligases join blunt-end and 3'-overhang-containing DNA substrates with similar catalytic efficiency, but LIG1 joins 5'-overhang-containing DNA substrates ∼20-fold less efficiently than LIG3 under optimal conditions. LIG1-catalyzed EJ is compromised at a physiological concentration of Mg2+, but its activity is restored by increased molecular crowding. In contrast to LIG1, LIG3 efficiently catalyzes EJ reactions at a physiological concentration of Mg2+ with or without molecular crowding. Under all tested conditions, LIG3 has greater affinity than LIG1 for DNA ends. Remarkably, LIG3 can ligate both strands of a DSB during a single binding encounter. The weaker DNA binding affinity of LIG1 causes significant abortive ligation that is sensitive to molecular crowding and DNA terminal structure. These results provide new insights into mechanisms of alternative nonhomologous EJ.

Mechanism of human Lig1 regulation by PCNA in Okazaki fragment sealing.

During lagging strand synthesis, DNA Ligase 1 (Lig1) cooperates with the sliding clamp PCNA to seal the nicks between Okazaki fragments generated by Pol δ and Flap endonuclease 1 (FEN1). We present several cryo-EM structures combined with functional assays, showing that human Lig1 recruits PCNA to nicked DNA using two PCNA-interacting motifs (PIPs) located at its disordered N-terminus (PIPN-term) and DNA binding domain (PIPDBD). Once Lig1 and PCNA assemble as two-stack rings encircling DNA, PIPN-term is released from PCNA and only PIPDBD is required for ligation to facilitate the substrate handoff from FEN1. Consistently, we observed that PCNA forms a defined complex with FEN1 and nicked DNA, and it recruits Lig1 to an unoccupied monomer creating a toolbelt that drives the transfer of DNA to Lig1. Collectively, our results provide a structural model on how PCNA regulates FEN1 and Lig1 during Okazaki fragments maturation.

Exploiting DNA Ligase III addiction of multiple myeloma by flavonoid Rhamnetin.

BACKGROUND: DNA ligases are crucial for DNA repair and cell replication since they catalyze the final steps in which DNA breaks are joined. DNA Ligase III (LIG3) exerts a pivotal role in Alternative-Non-Homologous End Joining Repair (Alt-NHEJ), an error-prone DNA repair pathway often up-regulated in genomically unstable cancer, such as Multiple Myeloma (MM). Based on the three-dimensional (3D) LIG3 structure, we performed a computational screening to identify LIG3-targeting natural compounds as potential candidates to counteract Alt-NHEJ activity in MM. METHODS: Virtual screening was conducted by interrogating the Phenol Explorer database. Validation of binding to LIG3 recombinant protein was performed by Saturation Transfer Difference (STD)-nuclear magnetic resonance (NMR) experiments. Cell viability was analyzed by Cell Titer-Glo assay; apoptosis was evaluated by flow cytometric analysis following Annexin V-7AAD staining. Alt-NHEJ repair modulation was evaluated using plasmid re-joining assay and Cytoscan HD. DNA Damage Response protein levels were analyzed by Western blot of whole and fractionated protein extracts and immunofluorescence analysis. The mitochondrial DNA (mtDNA) copy number was determined by qPCR. In vivo activity was evaluated in NOD-SCID mice subcutaneously engrafted with MM cells. RESULTS: Here, we provide evidence that a natural flavonoid Rhamnetin (RHM), selected by a computational approach, counteracts LIG3 activity and killed Alt-NHEJ-dependent MM cells. Indeed, Nuclear Magnetic Resonance (NMR) showed binding of RHM to LIG3 protein and functional experiments revealed that RHM interferes with LIG3-driven nuclear and mitochondrial DNA repair, leading to significant anti-MM activity in vitro and in vivo. CONCLUSION: Taken together, our findings provide proof of concept that RHM targets LIG3 addiction in MM and may represent therefore a novel promising anti-tumor natural agent to be investigated in an early clinical setting.

DNA damage and repair responses to freezing and anoxia stresses in wood frogs, Rana sylvatica.

Wood frogs, Rana sylvatica, endure the freezing of ∼65% of total body water while overwintering in cold climates, enduring not only internal ice formation but also long-term anoxia due to cessation of heartbeat and breathing. Thawing restores perfusion but rapid reoxygenation can increase vulnerability to reactive oxygen species and induce oxidative damage. This study provides a first assessment of antioxidant capacity, DNA damage, and DNA repair responses comparing freeze/thaw and anoxia/reoxygenation in liver and skeletal muscle of wood frogs. Oxidation of guanine resides in DNA did not change under either stress but total antioxidant capacity rose in both tissues under anoxia. Relative expression of eight proteins involved in double-stranded break repair (Mre11, Rad50, phospho-p95, XLF, DNA ligase IV, XRCC4, Ku70, Rad51) were assessed in both tissues. Freezing suppressed Ku70 and Rad51 in liver and Rad51 in muscle but levels rose again after thawing. Anoxia exposure suppressed XLF, Ku70 and Rad51 proteins in muscle. However, in liver, anoxia exposure led to elevated Mre11, Ku70 and DNA ligase IV, the former two belonging to the MRN complex that binds DNA and marks sites of double stranded breaks (DSBs). Large increases in Mre11 and Ku70 expression suggested DSB damage in liver under anoxia but not during freezing, whereas muscle was resistant to DSB damage under both stresses. These data indicate that DNA damage is minimal during whole body freezing due to tissue and stress specific regulation of antioxidant capacity and DNA damage repair to preserve genomic integrity.

Reconstitution of Mycobacterium marinum Nonhomologous DNA End Joining Pathway in Leishmania.

In mammalian cells, DNA double-strand breaks (DSBs) are mainly repaired by nonhomologous end joining (NHEJ) pathway. Ku (a heterodimer formed by Ku70 and Ku80 proteins) and DNA ligase IV are the core NHEJ factors. Ku could also be involved in other cellular processes, including telomere length regulation, DNA replication, transcription, and translation control. Leishmania, an early branching eukaryote and the causative agent of leishmaniasis, has no functional NHEJ pathway due to its lack of DNA ligase IV and other NHEJ factors but retains Ku70 and Ku80 proteins. In this study, we generated Leishmania donovani Ku70 disruption mutants and Ku70 and Ku80 double gene (Ku70/80) disruption mutants. We found that Leishmania Ku is still involved in DSB repair, possibly through its binding to DNA ends to block and slowdown 5' end resections and Ku-Ku or other protein interactions. Depending on location of a DSB between the direct repeat genomic sequences, Leishmania Ku could have an inhibiting effect, no effect or a promoting effect on the DSB repair mediated by single strand annealing (SSA), the most frequently used DSB repair pathway in Leishmania. Ku70/80 proteins are also required for the healthy proliferation of Leishmania cells. Interestingly, unlike in Trypanosoma brucei and L. mexicana, Ku70/80 proteins are dispensable for maintaining the normal lengths of telomeres in L. donovani. We also show it is possible to reconstitute the two components (Ku and Ligase D) NHEJ pathway derived from Mycobacterium marinum in Leishmania. This improved DSB repair fidelity and efficiency in Leishmania and sets up an example that the bacterial NHEJ pathway can be successfully reconstructed in an NHEJ-deficient eukaryotic parasite. IMPORTANCE Nonhomologous end joining (NHEJ) is the most efficient double-stranded DNA break (DSB) repair pathway in mammalian cells. In contrast, the protozoan parasite Leishmania has no functional NHEJ pathway but retains the core NHEJ factors of Ku70 and Ku80 proteins. In this study, we found that Leishmania Ku heterodimers are still participating in DSB repair possibly through blocking 5' end resections and Ku-Ku protein interactions. Depending on the DSB location, Ku could have an inhibiting or promoting effect on DSB repair mediated by the single-strand annealing repair pathway. Ku is also required for the normal growth of the parasite but surprisingly dispensable for maintaining the telomere lengths. Further, we show it is possible to introduce Mycobacterium marinum NHEJ pathway into Leishmania. Understanding DSB repair mechanisms of Leishmania may improve the CRISPR gene targeting specificity and efficiency and help identify new drug targets for this important human parasite.

Purification and Characterization of Human DNA Ligase IIIα Complexes After Expression in Insect Cells.

With improvements in biophysical approaches, there is growing interest in characterizing large, flexible multi-protein complexes. The use of recombinant baculoviruses to express heterologous genes in cultured insect cells has advantages for the expression of human protein complexes because of the ease of co-expressing multiple proteins in insect cells and the presence of a conserved post-translational machinery that introduces many of the same modifications found in human cells. Here we describe the preparation of recombinant baculoviruses expressing DNA ligase IIIα, XRCC1, and TDP1, their subsequent co-expression in cultured insect cells, the purification of complexes containing DNA ligase IIIα from insect cell lysates, and their characterization by multi-angle light scattering linked to size exclusion chromatography and negative stain electron microscopy.

DNA ligase and PCNA: Double-ring down to seal a break in DNA.

In this issue of Structure, Sverzhinsky et al. (2022) report structures of archaeal DNA ligase bound to the proliferating cell nuclear antigen (PCNA) sliding clamp and a nicked DNA substrate. The structures provide snapshots of ligation intermediates, which reveal a dynamic nature of the complex and explain how PCNA stimulates the DNA ligase activity.

DNA ligase IV mutations confer shorter lifespan and increased sensitivity to nutrient stress in Drosophila melanogaster.

The nonhomologous end-joining pathway is a primary DNA double-strand break repair pathway in eukaryotes. DNA ligase IV (Lig4) catalyzes the final step of DNA end ligation in this pathway. Partial loss of Lig4 in mammals causes Lig4 syndrome, while complete loss is embryonically lethal. DNA ligase 4 (DNAlig4) null Drosophila melanogaster is viable, but sensitive to ionizing radiation during early development. We proposed to explore if DNAlig4 loss induced other long-term sensitivities and defects in D. melanogaster. We demonstrated that DNAlig4 mutant strains had decreased lifespan and lower resistance to nutrient deprivation, indicating Lig4 is required for maintaining health and longevity in D. melanogaster.