DNA ligase

DNA ligase is an enzyme that stitches DNA strands together by creating phosphodiester bonds. In living organisms, it repairs breaks in one strand of the double helix, using the intact complementary strand as a guide to seal the final bond. Some versions, like DNA ligase IV, can also fix breaks in both strands. This enzyme is essential for DNA repair and replication, and it is widely used in labs for making recombinant DNA by joining DNA fragments.

The ligation process involves forming two phosphodiester bonds between a 3' hydroxyl end (the acceptor) and a 5' phosphate end (the donor). This uses two ATP molecules per bond and requires AMP. The reaction has four steps: first, the enzyme reorganizes around nicks or Okazaki fragments; second, it adds AMP to a lysine residue in its active site, releasing pyrophosphate; third, it transfers that AMP to the donor's 5' phosphate, creating a pyrophosphate bond; fourth, it forms the final phosphodiester bond between the donor's 5' phosphate and the acceptor's 3' hydroxyl. Ligase can also join blunt ends, but this needs higher enzyme concentrations and different conditions.

In *E. coli*, DNA ligase is encoded by the *lig* gene and uses energy from cleaving NAD to make bonds. It does not ligate blunt ends unless polyethylene glycol creates molecular crowding, and it cannot efficiently join RNA to DNA. Its activity can be boosted by DNA polymerase at low concentrations, but high polymerase levels hinder it.

The T4 bacteriophage ligase is the most common lab tool. It can join cohesive or blunt ends, oligonucleotides, RNA, and RNA-DNA hybrids, but not single-stranded nucleic acids. It works much better on blunt ends than *E. coli* ligase and requires ATP, not NAD. Engineered versions, like those fused to p50 or NF-kB, show over 160% more blunt-end activity. A typical reaction uses 0.01 units for sticky ends and 1 unit for blunt ends. Optimal temperature is 37°C, but 16°C is often used to balance activity with base-pairing. T4 ligase mutants are more sensitive to UV and alkylating agents, showing its role in repairing damage from these.

Mammals have four ligase types. DNA ligase 1 seals lagging-strand DNA after RNA primers are removed. DNA ligase 3 works with XRCC1 to seal DNA during nucleotide excision repair and is the only mammalian ligase found in mitochondria. DNA ligase 4 teams up with XRCC4 to finish non-homologous end joining for double-strand breaks and is needed for V(D)J recombination in immune system development. DNA ligase 2 is actually a degradation product of ligase 3, explaining the odd numbering. Eukaryotic and some microbial ligases use ATP instead of NAD.

Thermostable ligase, from a thermophilic bacterium, stays active at high temperatures—half-life of 48 hours at 65°C and over an hour at 95°C. It remains active through at least 500 thermal cycles, allowing very stringent hybridization and ligation.

Activity is measured in at least three units. The most common is the Weiss unit: the amount that exchanges 1 nmole of 32P from pyrophosphate to ATP in 20 minutes at 37°C. The rarely used Modrich-Lehman unit is the amount needed to convert 100 nmoles of d(A-T)n to an exonuclease-III resistant form in 30 minutes.