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.

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.

A role for the ATP-dependent DNA ligase lig E of Neisseria gonorrhoeae in biofilm formation.

BACKGROUND: The ATP-dependent DNA ligase Lig E is present as an accessory DNA ligase in numerous proteobacterial genomes, including many disease-causing species. Here we have constructed a genomic Lig E knock-out in the obligate human pathogen Neisseria gonorrhoeae and characterised its growth and infection phenotype. RESULTS: This demonstrates that N. gonorrhoeae Lig E is a non-essential gene and its deletion does not cause defects in replication or survival of DNA-damaging stressors. Knock-out strains were partially defective in biofilm formation on an artificial surface as well as adhesion to epithelial cells. In addition to in vivo characterisation, we have recombinantly expressed and assayed N. gonorrhoeae Lig E and determined the crystal structure of the enzyme-adenylate engaged with DNA substrate in an open non-catalytic conformation. CONCLUSIONS: These findings, coupled with the predicted extracellular/ periplasmic location of Lig E indicates a role in extracellular DNA joining as well as providing insight into the binding dynamics of these minimal DNA ligases.

Mammalian DNA ligases; roles in maintaining genome integrity.

The joining of breaks in the DNA phosphodiester backbone is essential for genome integrity. Breaks are generated during normal processes such as DNA replication, cytosine demethylation during differentiation, gene rearrangement in the immune system and germ cell development. In addition, they are generated either directly by a DNA damaging agent or indirectly due to damage excision during repair. Breaks are joined by a DNA ligase that catalyzes phosphodiester bond formation at DNA nicks with 3' hydroxyl and 5' phosphate termini. Three human genes encode ATP-dependent DNA ligases. These enzymes have a conserved catalytic core consisting of three subdomains that encircle nicked duplex DNA during ligation. The DNA ligases are targeted to different nuclear DNA transactions by specific protein-protein interactions. Both DNA ligase IIIα and DNA ligase IV form stable complexes with DNA repair proteins, XRCC1 and XRCC4, respectively. There is functional redundancy between DNA ligase I and DNA ligase IIIα in DNA replication, excision repair and single-strand break repair. Although DNA ligase IV is a core component of the major double-strand break repair pathway, non-homologous end joining, the other enzymes participate in minor, alternative double-strand break repair pathways. In contrast to the nucleus, only DNA ligase IIIα is present in mitochondria and is essential for maintaining the mitochondrial genome. Human immunodeficiency syndromes caused by mutations in either LIG1 or LIG4 have been described. Preclinical studies with DNA ligase inhibitors have identified potentially targetable abnormalities in cancer cells and evidence that DNA ligases are potential targets for cancer therapy.

Impact of polß/XRCC1 Interaction Variants on the Efficiency of Nick Sealing by DNA Ligase IIIα in the Base Excision Repair Pathway.

Base excision repair (BER) requires a coordination from gap filling by DNA polymerase (pol) ß to subsequent nick sealing by DNA ligase (LIG) IIIα at downstream steps of the repair pathway. X-ray cross-complementing protein 1 (XRCC1), a non-enzymatic scaffolding protein, forms repair complexes with polß and LIGIIIα. Yet, the impact of the polß mutations that affect XRCC1 interaction and protein stability on the repair pathway coordination during nick sealing by LIGIIIα remains unknown. Our results show that the polß colon cancer-associated variant T304 exhibits a reduced interaction with XRCC1 and the mutations in the interaction interface of V303 loop (L301R/V303R/V306R) and at the lysine residues (K206A/K244A) that prevent ubiquitin-mediated degradation of the protein exhibit a diminished repair protein complex formation with XRCC1. Furthermore, we demonstrate no significant effect on gap and nick DNA binding affinity of wild-type polß by these mutations. Finally, our results reveal that XRCC1 leads to an efficient channeling of nick repair products after nucleotide incorporation by polß variants to LIGIIIα, which is compromised by the L301R/V303R/V306R and K206A/K244A mutations. Overall, our findings provide insight into how the mutations in the polß/XRCC1 interface and the regions affecting protein stability could dictate accurate BER pathway coordination at the downstream steps involving nick sealing by LIGIIIα.

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.

The Contribution of DNA Ligase 4 Genetic Variations to Taiwanese Lung Cancer.

BACKGROUND/AIM: Impaired non-homologous end-joining DNA repair capacity may have a significant role in maintaining genome integrity and triggering carcinogenesis. However, the specific impact of DNA ligase 4 (Lig4) genotypes remains unclear. This study aimed to assess the contribution of Lig4 genotypes to the risk of developing lung cancer. MATERIALS AND METHODS: Polymerase chain reaction-restriction fragment length polymorphism analysis was used to examine the genotypes of Lig4 rs1805388, and their association with lung cancer risk was evaluated in a case-control study consisting of 358 lung cancer cases and 716 age- and sex-matched cancer-free control subjects. RESULTS: The distribution of CC, CT, and TT genotypes for Lig4 rs1805388 among the cases was 45.0%, 41.6%, and 13.4%, respectively, compared to 58.0%, 36.3%, and 5.7% among the controls (p for trend=1.98×10-6). Allelic analysis indicated that individuals carrying the T-allele for Lig4 rs1805388 had a 1.66-fold higher risk of developing lung cancer compared to those carrying the wild-type C-allele [95% confidence interval (CI)=1.36-2.02, p=4.04×10-7]. Moreover, a significant interaction was observed between the Lig4 rs1805388 genotype and smoking status (p=1.32×10-7). CONCLUSION: These findings suggest that the CT and TT variant genotypes of Lig4 rs1805388, combined with cigarette smoking, may contribute to a higher risk of developing lung cancer.

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.

Cryo-EM structure of a DNA-PK trimer: higher order oligomerisation in NHEJ.

The ability of humans to maintain the integrity of the genome is imperative for cellular survival. DNA double-strand breaks (DSBs) are considered the most critical type of DNA lesion, which can ultimately lead to diseases including cancer. Non-homologous end joining (NHEJ) is one of two core mechanisms utilized to repair DSBs. DNA-PK is a key component in this process and has recently been shown to form alternate long-range synaptic dimers. This has led to the proposal that these complexes can be formed before transitioning to a short-range synaptic complex. Here we present cryo-EM data representing an NHEJ supercomplex consisting of a trimer of DNA-PK in complex with XLF, XRCC4, and DNA Ligase IV. This trimer represents a complex of both long-range synaptic dimers. We discuss the potential role of the trimeric structure, and possible higher order oligomers, as structural intermediates in the NHEJ mechanism, or as functional DNA repair centers.

Autoimmunity and immunodeficiency associated with monoallelic LIG4 mutations via haploinsufficiency.

BACKGROUND: Biallelic mutations in LIG4 encoding DNA-ligase 4 cause a rare immunodeficiency syndrome manifesting as infant-onset life-threatening and/or opportunistic infections, skeletal malformations, radiosensitivity and neoplasia. LIG4 is pivotal during DNA repair and during V(D)J recombination as it performs the final DNA-break sealing step. OBJECTIVES: This study explored whether monoallelic LIG4 missense mutations may underlie immunodeficiency and autoimmunity with autosomal dominant inheritance. METHODS: Extensive flow-cytometric immune-phenotyping was performed. Rare variants of immune system genes were analyzed by whole exome sequencing. DNA repair functionality and T-cell-intrinsic DNA damage tolerance was tested with an ensemble of in vitro and in silico tools. Antigen-receptor diversity and autoimmune features were characterized by high-throughput sequencing and autoantibody arrays. Reconstitution of wild-type versus mutant LIG4 were performed in LIG4 knockout Jurkat T cells, and DNA damage tolerance was subsequently assessed. RESULTS: A novel heterozygous LIG4 loss-of-function mutation (p.R580Q), associated with a dominantly inherited familial immune-dysregulation consisting of autoimmune cytopenias, and in the index patient with lymphoproliferation, agammaglobulinemia, and adaptive immune cell infiltration into nonlymphoid organs. Immunophenotyping revealed reduced naive CD4+ T cells and low TCR-Vα7.2+ T cells, while T-/B-cell receptor repertoires showed only mild alterations. Cohort screening identified 2 other nonrelated patients with the monoallelic LIG4 mutation p.A842D recapitulating clinical and immune-phenotypic dysregulations observed in the index family and displaying T-cell-intrinsic DNA damage intolerance. Reconstitution experiments and molecular dynamics simulations categorize both missense mutations as loss-of-function and haploinsufficient. CONCLUSIONS: This study provides evidence that certain monoallelic LIG4 mutations may cause human immune dysregulation via haploinsufficiency.

Toxicity and therapy outcome associations in LIG3, SLCO1B3, ABCB1, OPRM1 and GSTP1 in high-grade serous ovarian cancer.

Adverse effects are the major limiting factors in combinatorial chemotherapies. To identify genetic associations in ovarian cancer chemotherapy-induced toxicities and therapy outcomes, we examined a cohort of 101 patients receiving carboplatin-paclitaxel treatment with advanced high-grade serous ovarian cancers. Based on literature and database searches, we selected 19 candidate polymorphisms, designed a multiplex single nucleotide polymorphism-genotyping assay and applied Cox regression analysis, case-control association statistics and the log-rank Mantel-Cox test. In the Cox regression analysis, the SLCO1B3 rs1052536 AA-genotype was associated with a reduced risk of any severe toxicity (hazard ratio = 0.35, p = 0.023). In chi-square allelic test, the LIG3 rs1052536 T-allele was associated with an increased risk of neuropathy (odds ratio [OR] = 2.79, p = 0.031) and GSTP1 rs1695 G allele with a poorer response in the first-line chemotherapy (OR = 2.65, p = 0.026). In Kaplan-Meier survival analysis, ABCB1 rs2032582 TT-genotype was associated with shorter overall survival (uncorrected p = 0.025) and OPRM1 rs544093 GG and GT genotypes with shorter platinum-free interval (uncorrected p = 0.027) and progression-free survival (uncorrected p = 0.012). Results suggest that SLCO1B3 and LIG3 variants are associated with the risk of adverse effects in patients receiving carboplatin-paclitaxel treatment, the GSTP1 variant may affect the treatment response and ABCB1 and OPRM1 variants may influence the prognosis.

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.

Profiling DNA Ligase Substrate Specificity with a Pacific Biosciences Single-Molecule Real-Time Sequencing Assay.

DNA ligases catalyze the joining of breaks in nucleic acid backbones and are essential enzymes for in vivo genome replication and repair across all domains of life. These enzymes are also critically important to in vitro manipulation of DNA in applications such as cloning, sequencing, and molecular diagnostics. DNA ligases generally catalyze the formation of a phosphodiester bond between an adjacent 5'-phosphate and 3'-hydroxyl in DNA, but they exhibit different substrate structure preferences, sequence-dependent biases in reaction kinetics, and variable tolerance for mismatched base pairs. Information on substrate structure and sequence specificity can inform both biological roles and molecular biology applications of these enzymes. Given the high complexity of DNA sequence space, testing DNA ligase substrate specificity on individual nucleic acid sequences in parallel rapidly becomes impractical when a large sequence space is investigated. Here, we describe methods for investigating DNA ligase sequence bias and mismatch discrimination using Pacific Biosciences Single-Molecule Real-Time (PacBio SMRT) sequencing technology. Through its rolling-circle amplification methodology, SMRT sequencing can give multiple reads of the same insert. This feature permits high-quality top- and bottom-strand consensus sequences to be determined while preserving information on top-bottom strand mismatches that can be obfuscated or lost when using other sequencing methods. Thus, PacBio SMRT sequencing is uniquely suited to measuring substrate bias and enzyme fidelity through multiplexing a diverse set of sequences in a single reaction. The protocols describe substrate synthesis, library preparation, and data analysis methods suitable for measuring fidelity and bias of DNA ligases. The methods are easily adapted to different nucleic acid substrate structures and can be used to characterize many enzymes under a variety of reaction conditions and sequence contexts in a rapid and high-throughput manner. © 2023 New England Biolabs and The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of overhang DNA substrates for ligation Basic Protocol 2: Preparation of ligation fidelity libraries Support Protocol 1: Preparation of ligation libraries for PacBio Sequel II sequencing Support Protocol 2: Loading and sequencing of a prepared library on the Sequel II instrument Basic Protocol 3: Computational processing of ligase fidelity sequencing data.

Human DNA polymerase η promotes RNA-templated error-free repair of DNA double-strand breaks.

A growing body of evidence indicates that RNA plays a critical role in orchestrating DNA double-strand break repair (DSBR). Recently, we showed that homologous nascent RNA can be used as a template for error-free repair of double-strand breaks (DSBs) in the transcribed genome and to restore the missing sequence at the break site via the transcription-coupled classical nonhomologous end-joining (TC-NHEJ) pathway. TC-NHEJ is a complex multistep process in which a reverse transcriptase (RT) is essential for synthesizing the DNA strand from template RNA. However, the identity of the RT involved in the TC-NHEJ pathway remained unknown. Here, we report that DNA polymerase eta (Pol η), known to possess RT activity, plays a critical role in TC-NHEJ. We found that Pol η forms a multiprotein complex with RNAP II and other TC-NHEJ factors, while also associating with nascent RNA. Moreover, purified Pol η, along with DSBR proteins PNKP, XRCC4, and Ligase IV can fully repair RNA templated 3'-phosphate-containing gapped DNA substrate. In addition, we demonstrate here that Pol η deficiency leads to accumulation of R-loops and persistent strand breaks in the transcribed genes. Finally, we determined that, in Pol η depleted but not in control cells, TC-NHEJ-mediated repair was severely abrogated when a reporter plasmid containing a DSB with several nucleotide deletion within the E. coli lacZ gene was introduced for repair in lacZ-expressing mammalian cells. Thus, our data strongly suggest that RT activity of Pol η is required in error-free DSBR.

Classical Recombinant DNA Cloning.

Traditional molecular cloning involves a series of linked experimental steps performed with the overall goal of isolating ("cloning") a specific DNA sequence-often a gene. The main purpose of cloning is to study either that DNA sequence or the RNA or protein product it encodes. Building on key enzymatic discoveries in the late 1960s, gene cloning was pioneered in the early 1970s. Since then, DNA cloning and manipulation have been used in every area of biological and biomedical research, from molecular genetics, structural biology, and developmental biology to neurobiology, ancient DNA studies, and immunology. It is a versatile technique that can be applied to a variety of starting DNA types and lengths, including cDNAs, genes, gene fragments, chromosomal regions, or shorter fragments such as PCR products and functional control regions such as enhancers or promoters. The starting DNA can originate from any cell, tissue, or organism. In this chapter we will cover traditional ("classic") molecular cloning strategy. This comprises six linked stages in which (1) PCR is used to amplify a DNA region of interest that is then (2) digested with restriction enzymes, alongside a selected vector, to produce complementary ends crucial for the two molecules to be (3) ligated by an ATP-dependent DNA ligase, creating a recombinant DNA molecule. The recombinant DNA is then (4) introduced into competent bacterial cells by transformation and (5) grown on a selective agar media, followed by (6) colony-PCR for screening purposes. We provide a worked example to demonstrate the cloning of an average-size gene (in this case the 2 kb DNA ligase A gene) from E. coli into a common plasmid expression vector. We have included six color figures and two tables to depict the key stages of a classical molecular cloning protocol. If you are cloning a segment of DNA or a gene, remember that each DNA cloning experiment is unique in terms of sequence, length, and experimental purpose. However, the principles of traditional cloning covered in this chapter are the same for any DNA sequence; we have included a detailed notes section, so you should easily be able to transfer them to your own work. Some of the following chapters in this volume will cover other, more recently developed, cloning protocols.

Assembling Multiple Fragments: The Gibson Assembly.

The Gibson Assembly is a popular method for molecular cloning which has been developed specifically to join several fragments together in a specific order, without the constraint of restriction enzyme sites. This method is based on the assembly of overlapping fragments, generally produced by PCR, and then combining them using three enzymes: a 5' exonuclease, a DNA polymerase, and a DNA ligase, in an isothermal reaction. Here, we describe this method, including the design of primers for the generation of the overlapping fragments and the assembly; to this end, we provide an example involving joining two fragments in a single plasmid.