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Literature summary extracted from

  • Maunders, M.J.
    DNA and RNA ligases (EC 6.5.1.1, EC 6.5.1.2, and EC 6.5.1.3) (1993), Methods Mol. Biol., 16, 213-230.
    View publication on PubMed

Activating Compound

EC Number Activating Compound Comment Organism Structure
6.5.1.1 Hexamine cobalt chloride maximal stimulation at 0.001-0.0015 mM, blunt end ligation is stimulated 50fold, but cohesive end ligation only 5fold Tequatrovirus T4
6.5.1.1 Polyethylene glycol polyethylene glycol 8000, maximal stimulation at 15% Tequatrovirus T4
6.5.1.1 Reducing agent reducing agents, e.g. 2-mercaptoethanol or dithiothreitol required Mammalia
6.5.1.1 Reducing agent reducing agents, e.g. 2-mercaptoethanol or dithiothreitol required Tequatrovirus T4
6.5.1.1 Reducing agent reducing agents, e.g. 2-mercaptoethanol or dithiothreitol required Escherichia phage T7
6.5.1.1 T4RNA ligase stimulates T4 DNA ligase activity Tequatrovirus T4
6.5.1.2 additional information no requirement for sulfhydryl reagent Escherichia coli

Application

EC Number Application Comment Organism
6.5.1.2 synthesis DNA ligase is used for cDNA cloning by replacement synthesis Escherichia coli

Cloned(Commentary)

EC Number Cloned (Comment) Organism
6.5.1.2
-
 Escherichia coli

General Stability

EC Number General Stability Organism
6.5.1.1 a 10 U/ml dilution loses 40% of its activity in 3 months Tequatrovirus T4

Inhibitors

EC Number Inhibitors Comment Organism Structure
6.5.1.1 Cs+ 0.2 M Tequatrovirus T4
6.5.1.1 dATP
-
 Escherichia phage T7
6.5.1.1 dATP
-
 Mammalia
6.5.1.1 dATP
-
 Tequatrovirus T4
6.5.1.1 K+ 0.2 M Tequatrovirus T4
6.5.1.1 Li+ 0.2 M Tequatrovirus T4
6.5.1.1 Na+ 0.2 M Tequatrovirus T4
6.5.1.1 NH4+ 0.2 M Tequatrovirus T4
6.5.1.1 phosphate inhibits blunt end ligation Tequatrovirus T4
6.5.1.1 spermidine
-
 Tequatrovirus T4
6.5.1.1 spermine
-
 Tequatrovirus T4
6.5.1.2 Mn2+ activation at 0.2-1.0 mM, inhibition at higher concentration Escherichia coli

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
6.5.1.2 0.000025 0.00056 5'-phosphate terminus of deoxyribonucleotides
-
 Salmonella enterica subsp. enterica serovar Typhimurium
6.5.1.2 0.000025 0.00056 5'-phosphate terminus of deoxyribonucleotides
-
 Bacillus subtilis
6.5.1.2 0.000025 0.00056 5'-phosphate terminus of deoxyribonucleotides
-
 Thermus thermophilus
6.5.1.2 0.000025 0.00056 5'-phosphate terminus of deoxyribonucleotides
-
 Escherichia coli
6.5.1.2 0.00003 0.00007 NAD+
-
 Salmonella enterica subsp. enterica serovar Typhimurium
6.5.1.2 0.00003 0.00007 NAD+
-
 Bacillus subtilis
6.5.1.2 0.00003 0.00007 NAD+
-
 Thermus thermophilus
6.5.1.2 0.00003 0.00007 NAD+
-
 Escherichia coli

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
6.5.1.1 Mg2+
-
 Mammalia
6.5.1.1 Mg2+ optimal concentration: 10 mM Tequatrovirus T4
6.5.1.1 Mn2+ 25% as effective as Mg2+ in activation Tequatrovirus T4
6.5.1.2 Ca2+ 60% as active as Mg2+ in activation as reported in one study, no activity in another Escherichia coli
6.5.1.2 K+
-
 Thermus thermophilus
6.5.1.2 K+ stimulates Escherichia coli
6.5.1.2 Mg2+ requires divalent cations, Mn2+ or Mg2+ Thermus thermophilus
6.5.1.2 Mg2+ requires divalent cations, Mn2+ or Mg2+ Escherichia coli
6.5.1.2 Mg2+ optimal concentration: 1-3 mM Escherichia coli
6.5.1.2 Mn2+ requires divalent cations, Mn2+ or Mg2+ Thermus thermophilus
6.5.1.2 Mn2+ requires divalent cations, Mn2+ or Mg2+ Escherichia coli
6.5.1.2 Mn2+ activation at 0.2-1.0 mM, inhibition at higher concentration Escherichia coli
6.5.1.2 NH4+
-
 Thermus thermophilus
6.5.1.2 NH4+ stimulates Escherichia coli
6.5.1.2 Rb+ stimulates Escherichia coli
6.5.1.2 Zn2+ slight activation Escherichia coli
6.5.1.3 Mg2+ required Tequatrovirus T4

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
6.5.1.1 63000
-
 1 * 63000, PAGE under denaturing and reducing conditions Tequatrovirus T4
6.5.1.1 68000
-
 gel filtration Tequatrovirus T4
6.5.1.2 74000
-
 1 * 74000, SDS-PAGE of denatured and reduced enzyme Escherichia coli
6.5.1.2 77000
-
 sedimentation equilibrium ultracentrifugation Escherichia coli
6.5.1.3 47000 48200 gel filtration, high-speed sedimentation equilibrium centrifugation Tequatrovirus T4

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
6.5.1.2 NAD+ + (deoxyribonucleotide)n + (deoxyribonucleotide)m Salmonella enterica subsp. enterica serovar Typhimurium joining of short DNA fragments formed during DNA replication and so enabling DNA synthesis to progress in an overall 3'-5' direction on the antiparallel strand of the double helix, while continual 5'-3' synthesis proceeds on the other strand. Plays a role during genetic recombination and in the repair of UV-damaged DNA ?
-
 ?
6.5.1.2 NAD+ + (deoxyribonucleotide)n + (deoxyribonucleotide)m Bacillus subtilis joining of short DNA fragments formed during DNA replication and so enabling DNA synthesis to progress in an overall 3'-5' direction on the antiparallel strand of the double helix, while continual 5'-3' synthesis proceeds on the other strand. Plays a role during genetic recombination and in the repair of UV-damaged DNA ?
-
 ?
6.5.1.2 NAD+ + (deoxyribonucleotide)n + (deoxyribonucleotide)m Thermus thermophilus joining of short DNA fragments formed during DNA replication and so enabling DNA synthesis to progress in an overall 3'-5' direction on the antiparallel strand of the double helix, while continual 5'-3' synthesis proceeds on the other strand. Plays a role during genetic recombination and in the repair of UV-damaged DNA ?
-
 ?
6.5.1.2 NAD+ + (deoxyribonucleotide)n + (deoxyribonucleotide)m Escherichia coli joining of short DNA fragments formed during DNA replication and so enabling DNA synthesis to progress in an overall 3'-5' direction on the antiparallel strand of the double helix, while continual 5'-3' synthesis proceeds on the other strand. Plays a role during genetic recombination and in the repair of UV-damaged DNA ?
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
6.5.1.1 Escherichia phage T7
-
-
-
6.5.1.1 Mammalia
-
-
-
6.5.1.1 Tequatrovirus T4
-
-
-
6.5.1.2 Bacillus subtilis
-
-
-
6.5.1.2 Escherichia coli
-
-
-
6.5.1.2 Salmonella enterica subsp. enterica serovar Typhimurium
-
-
-
6.5.1.2 Thermus thermophilus
-
-
-
6.5.1.3 Tequatrovirus T4
-
-
-

Storage Stability

EC Number Storage Stability Organism
6.5.1.1 -20°C, high concentrations of enzyme are very stable Tequatrovirus T4
6.5.1.1 -20°C, storage below -20°C is detrimental Tequatrovirus T4
6.5.1.3 -20°C Tequatrovirus T4

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
6.5.1.1 ATP + (deoxyribonucleotide)n + (deoxyribonucleotide)m
-
 Mammalia AMP + diphosphate + (deoxyribonucleotide)n+m
-
 ?
6.5.1.1 ATP + (deoxyribonucleotide)n + (deoxyribonucleotide)m
-
 Escherichia phage T7 AMP + diphosphate + (deoxyribonucleotide)n+m
-
 ?
6.5.1.1 ATP + (deoxyribonucleotide)n + (deoxyribonucleotide)m low DNA concentrations favor intramolecular reaction, i.e. recircularization, and higher concentrations favor intermolecular reaction, i.e. oligomerization and formation of recombinant molecules Tequatrovirus T4 AMP + diphosphate + (deoxyribonucleotide)n+m
-
 ?
6.5.1.1 ATP + (deoxyribonucleotide)n + (deoxyribonucleotide)m some activity in joining RNA molecules annealed to DNA and even RNA:RNA molecules Tequatrovirus T4 AMP + diphosphate + (deoxyribonucleotide)n+m
-
 ?
6.5.1.1 ATP + (deoxyribonucleotide)n + (deoxyribonucleotide)m catalyzes blunt end joining of DNA Tequatrovirus T4 AMP + diphosphate + (deoxyribonucleotide)n+m
-
 ?
6.5.1.1 ATP + (deoxyribonucleotide)n + (deoxyribonucleotide)m reverse reaction is catalyzed, the enzyme behaves as an AMP-dependent endonuclease, yielding nicked DNA Tequatrovirus T4 AMP + diphosphate + (deoxyribonucleotide)n+m
-
 ?
6.5.1.1 ATP + (deoxyribonucleotide)n + (deoxyribonucleotide)m ligation of flush-ended DNAs Tequatrovirus T4 AMP + diphosphate + (deoxyribonucleotide)n+m
-
 ?
6.5.1.1 additional information
-
 Mammalia ?
-
 ?
6.5.1.1 additional information ATP-diphosphate exchange reaction Tequatrovirus T4 ?
-
 ?
6.5.1.1 additional information ATP-diphosphate exchange reaction Escherichia phage T7 ?
-
 ?
6.5.1.2 ATP + 5'-phosphate terminus of deoxyribonucleotides
-
 Salmonella enterica subsp. enterica serovar Typhimurium ?
-
 ?
6.5.1.2 ATP + 5'-phosphate terminus of deoxyribonucleotides
-
 Bacillus subtilis ?
-
 ?
6.5.1.2 ATP + 5'-phosphate terminus of deoxyribonucleotides
-
 Thermus thermophilus ?
-
 ?
6.5.1.2 ATP + 5'-phosphate terminus of deoxyribonucleotides
-
 Escherichia coli ?
-
 ?
6.5.1.2 NAD+ + (deoxyribonucleotide)n + (deoxyribonucleotide)m DNA ligase requires two DNA termini, the 5'-terminus, carrying a phosphate group, and the 3'-terminus, a hydroxyl group. These termini must reside on a double-stranded molecule, DNA:DNA or DNA:RNA. Both strands of the duplex may terminate, in the form of a staggered end or a blunt end, and the ligase then requires a second similar double-stranded terminus to join the two in an intermolecular reaction. Alternatively, the two termini may be provided by a nick in just one strand of a duplex, which the enzyme will then seal Salmonella enterica subsp. enterica serovar Typhimurium AMP + nicotinamide nucleotide + (deoxyribonucleotide)n+m
-
 ?
6.5.1.2 NAD+ + (deoxyribonucleotide)n + (deoxyribonucleotide)m DNA ligase requires two DNA termini, the 5'-terminus, carrying a phosphate group, and the 3'-terminus, a hydroxyl group. These termini must reside on a double-stranded molecule, DNA:DNA or DNA:RNA. Both strands of the duplex may terminate, in the form of a staggered end or a blunt end, and the ligase then requires a second similar double-stranded terminus to join the two in an intermolecular reaction. Alternatively, the two termini may be provided by a nick in just one strand of a duplex, which the enzyme will then seal Bacillus subtilis AMP + nicotinamide nucleotide + (deoxyribonucleotide)n+m
-
 ?
6.5.1.2 NAD+ + (deoxyribonucleotide)n + (deoxyribonucleotide)m DNA ligase requires two DNA termini, the 5'-terminus, carrying a phosphate group, and the 3'-terminus, a hydroxyl group. These termini must reside on a double-stranded molecule, DNA:DNA or DNA:RNA. Both strands of the duplex may terminate, in the form of a staggered end or a blunt end, and the ligase then requires a second similar double-stranded terminus to join the two in an intermolecular reaction. Alternatively, the two termini may be provided by a nick in just one strand of a duplex, which the enzyme will then seal Thermus thermophilus AMP + nicotinamide nucleotide + (deoxyribonucleotide)n+m
-
 ?
6.5.1.2 NAD+ + (deoxyribonucleotide)n + (deoxyribonucleotide)m DNA ligase requires two DNA termini, the 5'-terminus, carrying a phosphate group, and the 3'-terminus, a hydroxyl group. These termini must reside on a double-stranded molecule, DNA:DNA or DNA:RNA. Both strands of the duplex may terminate, in the form of a staggered end or a blunt end, and the ligase then requires a second similar double-stranded terminus to join the two in an intermolecular reaction. Alternatively, the two termini may be provided by a nick in just one strand of a duplex, which the enzyme will then seal Escherichia coli AMP + nicotinamide nucleotide + (deoxyribonucleotide)n+m
-
 ?
6.5.1.2 NAD+ + (deoxyribonucleotide)n + (deoxyribonucleotide)m no ligation of blunt-ended or flush-ended DNAs Escherichia coli AMP + nicotinamide nucleotide + (deoxyribonucleotide)n+m
-
 ?
6.5.1.2 NAD+ + (deoxyribonucleotide)n + (deoxyribonucleotide)m joining of short DNA fragments formed during DNA replication and so enabling DNA synthesis to progress in an overall 3'-5' direction on the antiparallel strand of the double helix, while continual 5'-3' synthesis proceeds on the other strand. Plays a role during genetic recombination and in the repair of UV-damaged DNA Salmonella enterica subsp. enterica serovar Typhimurium ?
-
 ?
6.5.1.2 NAD+ + (deoxyribonucleotide)n + (deoxyribonucleotide)m joining of short DNA fragments formed during DNA replication and so enabling DNA synthesis to progress in an overall 3'-5' direction on the antiparallel strand of the double helix, while continual 5'-3' synthesis proceeds on the other strand. Plays a role during genetic recombination and in the repair of UV-damaged DNA Bacillus subtilis ?
-
 ?
6.5.1.2 NAD+ + (deoxyribonucleotide)n + (deoxyribonucleotide)m joining of short DNA fragments formed during DNA replication and so enabling DNA synthesis to progress in an overall 3'-5' direction on the antiparallel strand of the double helix, while continual 5'-3' synthesis proceeds on the other strand. Plays a role during genetic recombination and in the repair of UV-damaged DNA Thermus thermophilus ?
-
 ?
6.5.1.2 NAD+ + (deoxyribonucleotide)n + (deoxyribonucleotide)m joining of short DNA fragments formed during DNA replication and so enabling DNA synthesis to progress in an overall 3'-5' direction on the antiparallel strand of the double helix, while continual 5'-3' synthesis proceeds on the other strand. Plays a role during genetic recombination and in the repair of UV-damaged DNA Escherichia coli ?
-
 ?
6.5.1.3 ATP + (ribonucleotide)n + (ribonucleotide)m
-
 Tequatrovirus T4 AMP + diphosphate + (ribonucleotide)n+m
-
 ?
6.5.1.3 dATP + (ribonucleotide)n + (ribonucleotide)m acts on single-stranded or double-stranded DNA molecules. Acts on very small pieces of ribonucleic acid, with 40mers being the probable upper size limit. The minimum size of the 5'-moiety is a ribonucleoside 3',5'-bisphosphate Tequatrovirus T4 dAMP + phosphate + (ribonucleotide)n+m
-
 ?

Subunits

EC Number Subunits Comment Organism
6.5.1.1 monomer 1 * 63000, PAGE under denaturing and reducing conditions Tequatrovirus T4
6.5.1.2 monomer
-
 Thermus thermophilus
6.5.1.2 monomer 1 * 74000, SDS-PAGE of denatured and reduced enzyme Escherichia coli

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
6.5.1.1 4
-
 ligation of cohesive ends Tequatrovirus T4
6.5.1.1 37
-
 sealing nicks Tequatrovirus T4
6.5.1.2 10 15 ligation of cohesive ends Escherichia coli
6.5.1.2 24 37 cohesive-end DNA restriction fragments Thermus thermophilus
6.5.1.2 65 72 nick-closing activity Thermus thermophilus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
6.5.1.1 6.5
-
 ATP-diphosphate exchange reaction Tequatrovirus T4
6.5.1.1 7.2 7.8 joining of nicks in Tris-HCl buffer Tequatrovirus T4
6.5.1.1 7.5 8 joining of DNA Tequatrovirus T4
6.5.1.2 7.5 8 Tris-HCl buffer Escherichia coli
6.5.1.2 8
-
-
 Thermus thermophilus
6.5.1.2 8
-
 sodium phosphate buffer Escherichia coli

pH Range

EC Number pH Minimum pH Maximum Comment Organism
6.5.1.1 6.9 8.3 6.9: 40% of maximal activity, 8.3: 65% of maximal activity, joing of DNA Tequatrovirus T4
6.5.1.2 5.6 7.5 50% of maximal activity at pH 5.6 and 7.5, potassium phosphate buffer Escherichia coli