DNA Ligase Reaction: Mechanism and Biological Role
By Dr. Zubair Khalid, DVM, MS, PhD ·
Key Takeaways
- DNA ligase catalyzes the formation of phosphodiester bonds between adjacent nucleotides, requiring a 3′ hydroxyl and a 5′ phosphate group at a nick in double-stranded DNA. The reaction proceeds via three steps: enzyme adenylation, AMP transfer to the 5′ phosphate of DNA, and final phosphodiester bond formation with AMP release.
- Cofactor specificity dictates ligase function: ATP-dependent ligases are found in eukaryotes and bacteriophages, while NAD⁺-dependent ligases are characteristic of bacteria, making the latter potential targets for antibacterial drug development.
- The energy for phosphodiester bond formation is derived from the hydrolysis of the high-energy cofactor (ATP or NAD⁺), not from the DNA substrate itself, and requires the presence of divalent metal ions, typically Mg²⁺.
- Eukaryotic cells employ distinct DNA ligases for critical processes: Ligase I is essential for joining Okazaki fragments during lagging strand replication, Ligase III participates in base excision repair (BER) and nucleotide excision repair (NER), and Ligase IV is crucial for non-homologous end joining (NHEJ) of double-strand breaks.
- Blunt-end ligation, the joining of DNA fragments without complementary overhangs, is significantly less efficient than sticky-end ligation and requires high DNA concentrations and specific buffer conditions (e.g., 10 mM MgCl₂, 1 mM ATP for T4 DNA ligase) to promote end-to-end association.
Introduction to DNA Ligase and Its Reaction
What is DNA ligase?
DNA ligase is the enzyme responsible for catalyzing the formation of phosphodiester bonds between adjacent nucleotides in a DNA strand. It is the final actor in virtually every DNA transaction that requires strand joining: Okazaki fragment maturation during replication, the completion of almost all DNA repair pathways, and the end-joining steps of recombination. Without DNA ligase, the genome would fragment into pieces during every round of replication, and repair pathways would leave behind single-stranded gaps that are lethal to the cell.
The enzyme is found in all domains of life, from bacteria to humans, and is also encoded by bacteriophages such as T4 and T7. The T4 enzyme, in particular, has become a workhorse of molecular biology laboratories worldwide. For a broader overview of the enzyme's properties, see the DNA Ligase Definition.
DNA ligase is a member of the nucleotidyltransferase superfamily, a group of enzymes that use a nucleotide cofactor to activate a phosphate group before forming the new bond. The reaction it catalyzes is thermodynamically unfavorable on its own; the enzyme couples bond formation to the hydrolysis of a high-energy cofactor—either ATP or NAD⁺—to drive the reaction forward.
The overall reaction: phosphodiester bond formation
The DNA ligase reaction joins a 5′ phosphate group on one DNA strand to the 3′ hydroxyl group on an adjacent strand. The product is a continuous DNA backbone with a standard 3′–5′ phosphodiester linkage. The overall reaction can be written as:
DNA with a nick + ATP (or NAD⁺) → sealed DNA + AMP + PPi (or NMN + AMP)
The key point is that the energy for bond formation comes from cleavage of the high-energy phosphoanhydride bond in the cofactor, not from the DNA substrate itself. The reaction proceeds through three distinct chemical steps, each involving a covalent enzyme–nucleotide intermediate. Understanding these steps is essential for understanding how the enzyme achieves its specificity and why certain experimental conditions are required.
The Three-Step Mechanism of DNA Ligase
The DNA ligase reaction is a ping-pong mechanism with three discrete chemical steps. Each step involves the formation and breakdown of covalent intermediates, and each requires specific divalent metal ions, typically Mg²⁺.
Step 1: Enzyme adenylation
The first step is the activation of the enzyme itself. DNA ligase reacts with its cofactor—ATP in eukaryotes, archaea, and bacteriophages, or NAD⁺ in bacteria—to form a covalent enzyme–adenylate intermediate. In this reaction, the enzyme attacks the α-phosphate of ATP (or the AMP moiety of NAD⁺), releasing pyrophosphate (PPi) in the case of ATP, or nicotinamide mononucleotide (NMN) in the case of NAD⁺.
The adenylate group is transferred to a conserved lysine residue in the active site of the enzyme. This lysine is absolutely conserved across all DNA ligases and is part of the signature motif KxDGxR found in the nucleotidyltransferase superfamily. The covalent linkage is a phosphoramide bond between the ε-amino group of the lysine and the phosphate of AMP.
This step requires Mg²⁺, which coordinates the phosphate groups of the cofactor and stabilizes the transition state. The reaction is reversible in principle, but the release of PPi (or NMN) drives it forward under physiological conditions. In the absence of a DNA substrate, the enzyme remains in this adenylated state, primed and ready to transfer the AMP group to DNA.
Step 2: AMP transfer to DNA
The second step is the transfer of the AMP group from the enzyme to the DNA substrate. The enzyme binds to a nicked DNA molecule—a duplex in which one strand has a break between a 3′ hydroxyl and a 5′ phosphate. The enzyme recognizes this structure specifically; it does not bind efficiently to intact duplex DNA or to single-stranded DNA.
The adenylated enzyme positions the 5′ phosphate of the nick in its active site. The lysine–AMP phosphoramide bond is then attacked by the 5′ phosphate oxygen of the DNA, transferring the AMP group to the DNA. The product is a DNA molecule with a 5′–5′ pyrophosphate linkage: the 5′ phosphate of the DNA is now adenylated (AppDNA). The enzyme is now deadenylylated and no longer covalently linked to AMP.
This step is also Mg²⁺-dependent. The metal ion coordinates the phosphate groups and stabilizes the developing negative charge during the nucleophilic attack. The enzyme undergoes a conformational change upon DNA binding that positions the 5′ phosphate precisely for attack on the lysine–AMP bond.
Step 3: Sealing the nick
The final step is the attack of the 3′ hydroxyl group of the nick on the activated 5′ phosphate. The 3′ OH of the downstream DNA strand performs a nucleophilic attack on the phosphorus atom of the adenylated 5′ phosphate. This displaces AMP and forms the new phosphodiester bond, sealing the nick.
The leaving group is AMP, which is released from the DNA and diffuses away. The enzyme is now free to bind another cofactor molecule and begin a new catalytic cycle. The overall reaction has consumed one molecule of ATP (or NAD⁺) and produced one molecule of AMP and one molecule of PPi (or NMN).
The third step is the rate-limiting step of the reaction under most conditions. It requires proper alignment of the 3′ OH and the adenylated 5′ phosphate, which is achieved by the enzyme's ability to distort the DNA duplex and bring the two ends into close proximity. The reaction is highly specific for a 3′ OH group; a 3′ phosphate or a 3′ dideoxy terminus cannot serve as the nucleophile.
Cofactor Requirements: NAD+ vs ATP
DNA ligases are classified into two families based on the cofactor they use. This distinction is evolutionarily ancient and has practical implications for drug development and experimental design.
NAD+-dependent ligases
NAD⁺-dependent DNA ligases are found in bacteria and some archaea. They are absent from eukaryotes, which makes them attractive targets for antibacterial drug development. The enzyme uses NAD⁺ as the adenylate donor, cleaving the molecule between the NMN and AMP moieties. The products are enzyme–AMP and NMN.
The NAD⁺-dependent ligase (encoded by the ligA gene in E. coli) is essential for bacterial viability. It is a larger protein than its ATP-dependent counterparts, with additional domains involved in NAD⁺ binding and in interaction with other replication proteins. The enzyme is zinc-dependent as well as magnesium-dependent; a zinc-binding domain is required for full activity.
The reaction chemistry is identical to that of ATP-dependent ligases after the initial adenylation step. The difference is only in how the enzyme–AMP intermediate is generated. NAD⁺-dependent ligases cannot use ATP, and ATP-dependent ligases cannot use NAD⁺. This strict specificity is determined by the structure of the cofactor-binding pocket in each enzyme.
ATP-dependent ligases
ATP-dependent DNA ligases are found in eukaryotes, archaea, and bacteriophages. They use ATP as the adenylate donor, releasing pyrophosphate in the first step. The eukaryotic nucleus contains three major ATP-dependent ligases—Ligase I, Ligase III, and Ligase IV—each with distinct roles in replication and repair. A fourth, Ligase II, is a proteolytic fragment of Ligase III and is not a distinct gene product.
The T4 DNA ligase, encoded by bacteriophage T4 gene 30, is the most widely used ATP-dependent ligase in molecular biology. It is a relatively small protein (~55 kDa) that is remarkably efficient at joining both nicked DNA and blunt-ended fragments. Its properties are discussed further in the context of DNA Ligase in Genetic Engineering.
ATP-dependent ligases share a conserved catalytic core with NAD⁺-dependent ligases, despite their different cofactor usage. The core domain contains the active-site lysine and the nucleotide-binding pocket. The cofactor specificity is determined by accessory domains that recognize either ATP or NAD⁺ specifically. For more detail on the ATP requirement, see DNA Ligase Use ATP.
Substrate Specificity and Reaction Conditions
Nicked DNA vs double-strand breaks
DNA ligase is highly specific for its substrate. The preferred substrate is a nick in double-stranded DNA: a site where the phosphodiester backbone is interrupted but the two strands remain base-paired. The enzyme recognizes the duplex structure and the precise geometry of the 3′ OH and 5′ phosphate at the nick.
The enzyme does not ligate RNA–DNA junctions efficiently, nor does it act on single-stranded DNA. It requires a double-stranded region of at least a few base pairs on either side of the nick for stable binding. The minimal substrate is approximately 4–6 base pairs of duplex DNA flanking the nick.
Double-strand breaks are a different matter. A double-strand break involves a complete break in both strands of the duplex, with the two ends no longer held together by base pairing. DNA ligase cannot simply join two free ends in solution; it requires the ends to be brought into close proximity and held in the correct orientation. In the cell, this is achieved by accessory proteins that tether the two ends together. In the test tube, high DNA concentrations and macromolecular crowding agents (such as PEG) are used to promote end-to-end association.
Blunt-end ligation and its inefficiency
Blunt-end ligation is the joining of two DNA molecules that have no complementary overhangs. The 3′ OH and 5′ phosphate are present on the same molecule but are not adjacent; they must be brought together by collision of two separate DNA molecules. This is a bimolecular reaction and is therefore concentration-dependent.
The efficiency of blunt-end ligation is much lower than that of sticky-end ligation. The rate constant for blunt-end joining is typically 100–1000-fold lower than for cohesive-end joining. This is because the enzyme must bind to the end of one DNA molecule, then capture a second DNA molecule and align the two ends precisely. The reaction is second-order with respect to DNA concentration, meaning that doubling the DNA concentration quadruples the rate of blunt-end ligation.
In practice, blunt-end ligation is performed at high DNA concentrations (typically 1–10 μg/mL for vector ligation) and at low temperatures (4–16 °C) to stabilize the transient end-to-end associations. T4 DNA ligase is the enzyme of choice for blunt-end ligation because it is more efficient at this reaction than E. coli ligase. The DNA Ligase Join Sticky resource provides additional detail on sticky-end versus blunt-end considerations.
The standard reaction buffer for T4 DNA ligase contains 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 10 mM DTT, and 1 mM ATP. The Mg²⁺ is essential for catalysis; it coordinates the phosphate groups in both the cofactor and the DNA substrate. The pH optimum is 7.5–8.0, reflecting the need for a deprotonated 3′ OH group for the final nucleophilic attack. DTT is included to maintain the enzyme's cysteine residues in a reduced state.
Methods to Study DNA Ligase Activity
In vitro ligation assays
The simplest assay for DNA ligase activity is the conversion of a nicked plasmid to a closed circular form. A plasmid with a single nick is incubated with DNA ligase and cofactor, and the reaction products are analyzed by agarose gel electrophoresis. The nicked form (relaxed circle) migrates more slowly than the closed circular form (supercoiled), so successful ligation is detected as a shift in mobility.
This assay is quantitative if the DNA is radiolabeled or stained with a fluorescent dye. The fraction of closed circular product can be measured by densitometry, and the reaction rate can be determined from the time course. The assay is simple, robust, and requires only standard laboratory equipment.
A more sensitive assay uses a synthetic oligonucleotide substrate with a fluorescent label at one end. The substrate is a short duplex (typically 30–50 base pairs) with a single nick in one strand. Ligation is detected by denaturing polyacrylamide gel electrophoresis, which separates the nicked substrate from the full-length product. This assay allows precise measurement of reaction rates and is suitable for kinetic analysis.
Detecting ligation products
Radioactive labeling remains a gold standard for detecting ligation products. A 5′ phosphate on the DNA is labeled with ³²P using T4 polynucleotide kinase, and the ligation product is detected by autoradiography after gel electrophoresis. The nicked substrate and the ligated product differ in length by one nucleotide, so they are readily separated on a denaturing gel.
Fluorescence-based methods have largely replaced radioactive methods in modern laboratories. A fluorophore can be attached to the 5′ end of one oligonucleotide and a quencher to the 3′ end of the adjacent oligonucleotide. When the nick is sealed, the fluorophore and quencher are brought into proximity, and the fluorescence signal decreases. This assay is amenable to real-time monitoring in a plate reader and is used for high-throughput screening of ligase inhibitors.
A third approach is the use of molecular beacons or FRET-based probes that change their fluorescence upon ligation. These assays are highly sensitive and can detect femtomole quantities of product. They are particularly useful for studying ligase kinetics under a wide range of conditions.
Biological Roles in DNA Replication and Repair
Ligase I in replication
DNA ligase I is the major replicative ligase in eukaryotic cells. It is responsible for joining Okazaki fragments on the lagging strand during DNA replication. Okazaki fragments are short (~100–200 nucleotides in eukaryotes) pieces of DNA synthesized discontinuously in the 5′ to 3′ direction. Each fragment begins with an RNA primer that must be removed and replaced with DNA before the fragments can be joined.
The joining of Okazaki fragments requires the coordinated action of several enzymes. The RNA primer is removed by RNase H and FEN1 (flap endonuclease 1), which cleave the RNA and any displaced DNA flap. DNA polymerase δ then fills the gap, and DNA ligase I seals the remaining nick. Ligase I is recruited to the replication fork through its interaction with PCNA (proliferating cell nuclear antigen), the sliding clamp that processivity factor for replicative polymerases.
Ligase I is a 102 kDa protein with a conserved catalytic domain at the C-terminus and a regulatory N-terminal domain. The N-terminal domain contains a nuclear localization signal and a PCNA-binding motif. Mutations in LIG1 cause a rare human syndrome characterized by growth retardation, immunodeficiency, and increased cancer susceptibility, underscoring the essential role of this enzyme in maintaining genomic stability.
Ligase III in repair
DNA ligase III is the major ligase in base excision repair (BER) and also participates in nucleotide excision repair (NER) and homologous recombination. It exists in two isoforms: Ligase IIIα, which is nuclear, and Ligase IIIβ, which is mitochondrial. The nuclear form contains a zinc finger domain at its N-terminus that binds to nicked DNA with high affinity.
Ligase IIIα forms a stable complex with XRCC1 (X-ray repair cross-complementing protein 1), a scaffold protein that coordinates the activities of several BER enzymes. The Ligase III–XRCC1 complex is recruited to sites of DNA damage by poly(ADP-ribose) polymerase 1 (PARP1), which binds to single-strand breaks and synthesizes poly(ADP-ribose) chains that serve as a recruitment signal.
In BER, the damaged base is removed by a DNA glycosylase, the resulting abasic site is cleaved by AP endonuclease 1 (APE1), and the gap is filled by DNA polymerase β. DNA ligase III then seals the nick, completing the repair. This pathway is essential for repairing the thousands of spontaneous base lesions that occur in every cell each day.
Ligase IV in non-homologous end joining
DNA ligase IV is the ligase responsible for non-homologous end joining (NHEJ), the major pathway for repairing double-strand breaks in mammalian cells. NHEJ is a error-prone process that directly rejoins the two broken ends without requiring a homologous template. It is essential for V(D)J recombination, the process that generates antibody diversity in B cells and T cell receptor diversity in T cells.
Ligase IV is a 96 kDa protein that forms a tight complex with XRCC4, a scaffold protein that stabilizes the ligase and recruits it to DNA ends. The Ligase IV–XRCC4 complex is recruited to double-strand breaks by the Ku70/Ku80 heterodimer, which binds to DNA ends and recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The kinase phosphorylates several targets, including the ligase complex itself, and promotes end processing and ligation.
Ligase IV is unique among the eukaryotic ligases in its ability to ligate non-complementary ends. It can join ends with short microhomologies (1–4 base pairs) and can even ligate ends with no homology at all, albeit with low efficiency. This flexibility is essential for NHEJ, which must handle a wide variety of broken ends generated by ionizing radiation and other DNA-damaging agents. Mutations in LIG4 cause a severe combined immunodeficiency syndrome (LIG4 syndrome) characterized by radiation sensitivity, developmental delay, and immunodeficiency.
Common Pitfalls and Misconceptions
Energy source confusion
A common error is to think that DNA ligase uses the energy from ATP hydrolysis directly to form the phosphodiester bond. In fact, the energy is used to activate the 5′ phosphate through adenylation. The ATP is cleaved in the first step of the reaction, releasing PPi, and the AMP is transferred to the enzyme and then to the DNA. The final bond formation step does not involve ATP directly; it involves the attack of the 3′ OH on the adenylated 5′ phosphate.
Another related misconception is that the reaction consumes ATP and produces ADP. In fact, the products are AMP and PPi, not ADP and Pi. The cleavage of ATP occurs between the α and β phosphates, not between the β and γ phosphates. This is a distinctive feature of the nucleotidyltransferase mechanism.
Ligase vs polymerase
Students sometimes confuse DNA ligase with DNA polymerase. The two enzymes are fundamentally different. DNA polymerase synthesizes new DNA by adding nucleotides to a growing chain, using a template strand to direct base pairing. It requires a primer with a free 3′ OH and incorporates deoxyribonucleoside triphosphates (dNTPs), releasing pyrophosphate for each nucleotide added.
DNA ligase, by contrast, does not synthesize DNA. It joins two pre-existing DNA strands by forming a phosphodiester bond between a 3′ OH and a 5′ phosphate. It does not require a template, does not incorporate nucleotides, and does not read genetic information. The DNA Ligase Short resource provides a concise comparison.
A useful way to remember the difference: polymerase extends a strand, ligase seals a break. Polymerase creates new phosphodiester bonds between a nucleotide and the growing chain; ligase creates a phosphodiester bond between two existing chains.
Cofactor specificity
Another common error is to assume that all DNA ligases use ATP. In fact, bacterial ligases use NAD⁺, while eukaryotic and phage ligases use ATP. This distinction is not merely academic; it has practical implications. If you are performing a ligation with T4 DNA ligase (ATP-dependent) but your buffer contains NAD⁺ instead of ATP, the reaction will not proceed.
The cofactor specificity is absolute. An ATP-dependent ligase cannot use NAD⁺, and an NAD⁺-dependent ligase cannot use ATP. This is because the cofactor-binding pocket is shaped to accommodate the specific cofactor, and the enzyme must recognize the entire molecule, not just the AMP moiety.
Practical Summary and Exam Tips
How to approach exam questions
When answering exam questions about DNA ligase, always start by identifying the cofactor (ATP or NAD⁺) and the three steps of the mechanism. Draw the reaction if asked to explain it. Remember that the energy for bond formation comes from the cofactor, not from the DNA.
For questions about biological roles, be specific: Ligase I for Okazaki fragments, Ligase III for BER, Ligase IV for NHEJ. Mention the accessory proteins (PCNA, XRCC1, XRCC4) that recruit and regulate each ligase.
For experimental questions, remember the key conditions: pH 7.5–8.0, Mg²⁺ (10 mM), ATP (1 mM) for T4 ligase, and the need for high DNA concentrations for blunt-end ligation. The Thermo T4 DNA Ligase page provides additional practical details.
Frequently Asked Questions
What is the DNA ligase reaction mechanism?
The DNA ligase reaction proceeds in three steps. First, the enzyme reacts with ATP (or NAD⁺) to form a covalent enzyme–AMP intermediate, releasing PPi (or NMN). Second, the AMP is transferred to the 5′ phosphate of the DNA at a nick, forming an adenylated DNA intermediate. Third, the 3′ OH of the adjacent nucleotide attacks the adenylated 5′ phosphate, forming the phosphodiester bond and releasing AMP. Each step requires Mg²⁺.
Does DNA ligase require ATP?
It depends on the enzyme. Eukaryotic, archaeal, and bacteriophage ligases use ATP. Bacterial ligases use NAD⁺. The T4 DNA ligase used in molecular biology is ATP-dependent and requires ATP in the reaction buffer at a concentration of approximately 1 mM. The DNA Ligase Use ATP page provides further detail.
What is the role of AMP in DNA ligase reaction?
AMP is the adenylate group that is transferred from the cofactor to the enzyme and then to the DNA. It activates the 5′ phosphate of the DNA, making it a good leaving group for the final nucleophilic attack by the 3′ OH. AMP is released as a product of the reaction and can be recycled by the cell.
Why does DNA ligase need a nick?
A nick provides the precise geometry required for catalysis. The 3′ OH and 5′ phosphate must be positioned adjacent to each other in the enzyme's active site. A nick in double-stranded DNA presents these groups in the correct orientation. The enzyme does not ligate single-stranded DNA because the ends are not held in the proper position. The DNA Ligase Form Hydrogen Bonds resource explains the structural basis for substrate recognition.
What is the difference between DNA ligase and DNA polymerase?
DNA polymerase synthesizes new DNA by adding nucleotides to a primer, using a template strand. It requires dNTPs and releases pyrophosphate. DNA ligase joins two pre-existing DNA strands by forming a phosphodiester bond between a 3′ OH and a 5′ phosphate. It does not synthesize DNA and does not require a template. Polymerase extends, ligase seals.
How does DNA ligase work in DNA replication?
During DNA replication, DNA ligase I joins Okazaki fragments on the lagging strand. After the RNA primers are removed and the gaps are filled by DNA polymerase δ, ligase I seals the remaining nicks. This produces a continuous daughter strand. Ligase I is recruited to the replication fork through its interaction with PCNA.
What happens if DNA ligase is defective?
Defective DNA ligase leads to accumulation of DNA breaks and gaps. In bacteria, loss of NAD⁺-dependent ligase is lethal. In humans, mutations in LIG1 cause growth retardation, immunodeficiency, and cancer susceptibility. Mutations in LIG4 cause LIG4 syndrome, characterized by severe combined immunodeficiency, radiation sensitivity, and developmental abnormalities. In all cases, the inability to seal DNA breaks results in genomic instability and cell death.
Key Takeaways
- DNA ligase catalyzes phosphodiester bond formation between a 3′ OH and a 5′ phosphate at a nick in double-stranded DNA.
- The reaction has three steps: enzyme adenylation, AMP transfer to DNA, and phosphodiester bond formation with AMP release.
- ATP is the cofactor for eukaryotic and phage ligases; NAD⁺ is the cofactor for bacterial ligases.
- The energy for bond formation comes from cleavage of the cofactor, not from the DNA substrate.
- DNA ligase does not synthesize DNA; it joins pre-existing strands. DNA polymerase synthesizes, DNA ligase seals.
- Eukaryotic cells have three major ligases: Ligase I (replication), Ligase III (BER), and Ligase IV (NHEJ).
- Blunt-end ligation is inefficient and requires high DNA concentrations and low temperatures; sticky-end ligation is much more efficient.
- The reaction requires Mg²⁺ and a pH of 7.5–8.0; T4 DNA ligase buffers typically contain 10 mM MgCl₂ and 1 mM ATP.
Further Reading
- Pang L et al. DNA point mutation detection based on DNA ligase reaction and nano-Au amplification: a piezoelectric approach. Analytical biochemistry. 2006. PubMed 16996020
- Wang H et al. Combination of DNA ligase reaction and gold nanoparticle-quenched fluorescent oligonucleotides: a simple and efficient approach for fluorescent assaying of single-nucleotide polymorphisms. Analytical chemistry. 2010. PubMed 20726510
- Feng K et al. QCM detection of DNA targets with single-base mutation based on DNA ligase reaction and biocatalyzed deposition amplification. Biosensors & bioelectronics. 2007. PubMed 16963256
- Okahata Y et al. Quantitative detection of a DNA ligase reaction on a quartz-crystal microbalance. Nucleic acids symposium series. 1999. PubMed 10780422
- Little JW et al. Enzymatic joining of DNA strands, II. An enzyme-adenylate intermediate in the dpn-dependent DNA ligase reaction. Proceedings of the National Academy of Sciences of the United States of America. 1967. PubMed 4295585
- Shimer GH Jr, Backman KC. Ligase chain reaction. Methods in molecular biology (Clifton, N.J.). 1995. PubMed 7550715