DNA ligase

DNA ligase is a ligase-type enzyme that forms covalent bonds between the 5' end of one polynucleotide chain and the 3' end of another polynucleotide chain. Also called polynucleotide binding enzyme.

Excision repair is carried out by 3 enzymes: U.V. running nuclease, which initiates the process, DNA polymerase I, which eliminates and rebuilds the DNA fragment, and DNA ligase, which joins the new and old fragments.

To achieve a careful replication of DNA (deoxyribonucleic acid), each strand of the double helix acts as a template to synthesize the new strand that will have a complementary sequence to the template strand. On the one hand, as the two chains of the double helix have opposite directions (one is 5'-3' while the other is 3'-5') this process, which seems simple, is complicates as it needs specific mechanisms to synthesize the two complementary strands also in opposite directions.

On the other hand, in the double helix the chains are joined by their nitrogenous bases and therefore a complementary chain must be made to each already existing chain. First they have to be separated, then the complementary strand is created and finally each template and its complement must be wound around each other again to form the new DNA strand. At this moment, DNA ligases come into action to join the ends of this new chain.

What is DNA ligase?

The union of the Okazaki fragments (these are the complementary strands originated from the template strands) with the complementary strands requires an enzyme to catalyze it. This enzyme is DNA ligase.

Its function is to catalyze a phosphodiester bond between the 3'-hydroxyl group at the end of one of the DNA chains and the 5'-diphosphate group at the end of the other DNA chain. To carry out this reaction, the cell needs energy because it is a thermodynamically unfavorable reaction, that is, it is not a reaction that occurs spontaneously.

On the one hand, in eukaryotic cells and in archaea, the energy source is ATP (Adenosine Triphosphate). The ATP molecule is separated into AMP (Adenosine Monophosphate) and pyrophosphate to facilitate handling.

On the other hand, bacteria obtain energy from NAD+ (nicotinamide adenine dinucleotide) which is divided into AMP and NMN (nicotinamide mononucleotide) with the same purpose as ATP in eukaryotes and archaea.

DNA ligase cannot join two single-stranded DNA molecules (single-stranded DNA) or form circular single-stranded DNA, but rather seals breaks that have occurred in double-stranded DNA molecules. strand (DNA with two strands twisted into a double helix) at the time of separation of the double helix.

Escherichia coli bacteria are able to form a phosphodiester bond if there are at least a few nitrogenous bases of single-stranded DNA with the end of a double-stranded DNA fragment and form base pairs.

The ligase encoded by bacteriophage T4 can join two blunt-ended double helix fragments, this ability is exploited in recombinant DNA technology.

The simplest form of DNA is the one with a blunt end. These types of molecules end with a base pair. Blunt-ended molecules have two disadvantages; The first is that blunt-ended strands have less yield, and the second is that you have a better chance of inserting the desired DNA fragment in the opposite direction from the one you want. on the other hand, two blunt ends will always be compatible with each other.

The enzyme reacts with ATP or NAD+ to form an AMP molecule linked by a phosphoamide group with the amino group of the active site of lysine.B. DNA phosphate 50 attacks AMP phosphoryl group to form adenylated DNA.C. The ligase DNA catalyzes the reaction between the OH group of DNA and the activated phosphate 50 to form a fosphodiéster link thus freeing AMP.

Mechanism of action of ligases

To form the two covalent phosphodiester bonds between both ends of the two chains, DNA ligase catalyzes a reaction together with ATP that follows 3 steps:

1-Adenization of a residue in the active center of the enzyme releasing phosphate.

2- Transfer from AMP to phosphate 5 'from the donor originating the formation of a pyrophosphate link.

3-Formation of a phosphodiéster link between phosphate 5 ' of the donor and hydroxyl 3' of the acceptor.

When ligase works on broken ends, it requires a higher concentration of enzymes and different reaction conditions.

History

In 1967, scientists from various laboratories discovered DNA ligase. They were originally discovered in the T4 bacteriophage, Escherichia coli bacterium, as well as other bacteria.

Types of DNA ligases

There are two main branches of DNA ligases: the ATP-dependent and the NAD+-dependent DNA ligases. The ATP cofactor is the main one in mammalian cells while bacteria mainly use NAD+ as a cofactor for the enzyme, although there are bacteria with the ability to form enzymes that need ATP as a cofactor to function. However, both enzymes follow the same reactive mechanism.

DNA ligases in bacteria

DNA ligase from E.coli bacteria is encoded by the LIG gene. As in many prokaryotic organisms, the DNA ligase of this bacterium uses the energy created with the NAD cofactor to form phosphodiester bonds. This ligase cannot join the blunt ends of DNA except under certain molecular conditions with a significant presence of polyethylene glycol. It also cannot efficiently join RNA to DNA. This enzyme is used in laboratories to clone DNA molecules from dsDNa viruses.

DNA ligases in mammals

In mammals, up to 4 classes of DNA ligases can be determined whose main substrate is ATP. Three identified genes encode these DNA ligases: LIG1, LIG3, and LIG4. DNA ligase II, being a derivative of DNA ligase III (by proteolytic mechanism), reduces the number of genes involved in the biogenesis of these enzymes to 3 genes. The role played by each of these enzymes is determined by the proteins with which it interacts.

DNA ligase I

This DNA ligase is central to the mechanism of DNA replication. The active center of this enzyme is located in the domain of the non-catalytic amino terminal group. In addition, DNA ligase I participates in the excision of DNA, thus allowing its repair and maintenance together with other proteins such as beta DNA polymerase, with which DNA ligase I interacts directly, thus forming a multiprotein complex performing said function.

DNA ligase II

This enzyme is a fragment generated by proteolysis of DNA ligase III.

DNA ligase III

Two forms of DNA ligase III are generated by the LIG3 gene: alpha-DNA ligase III and beta-DNA ligase III. These enzymes differ in their ability to bind to the DNA repair protein, XRCC1. The XRCC1 protein only binds to alpha-DNA ligase and is done through the BRCT domain in the C-terminal fragment of the ligase polypeptides. This protein enables single-strand DNA damage repair and base excision repair (BER). On the other hand, beta-DNA ligase III seems to help in the completion of meiotic recombination or in postmeiosis DNA repair.

DNA ligase IV

DNA ligase IV binds to a DNA repair protein, XRCC4. This linkage is made from the C-terminal region of DNA ligase IV, which contains two BRCT domains. This interaction, stimulatory for DNA splicing activity, implies that this enzyme functions in the V(D)J genetic recombination system and in non-homologous end splicing of double-stranded DNA breaks.

Applications

Applications in Molecular Biology Research

Currently, DNA ligases are an indispensable tool in research, in the field of molecular biology, which is used to generate recombinant DNA sequences.

They are mainly used as restriction enzymes to insert DNA fragments almost always into genes from plasmid DNA molecules, although they can also be inserted into others.

To carry out this type of experiment, the temperature must be controlled above all. Since most experiments use T4 DNA ligases, they are performed at a temperature of 25 °C since this is the temperature at which the enzymatic activity of these proteins is highest. This temperature must be balanced with the optimal melting temperature for both chains to join. The most effective ligation temperature for blunt ends is 14-20°C.

Participation of DNA ligase in cloning processes

For the cloning of the DNA strands, plasmid vectors are used, small circular double-stranded molecules, derived from large plasmids (circular or linear extrachromosomal DNA molecules that replicate and transcribe independently of chromosomal DNA) that occur naturally. in almost all bacteria and certain yeasts. As a general rule they represent a small fragment of the total DNA of the host cell but can be separated due to their small size with respect to the larger chromosomal DNA molecules and that are pelleted by centrifugation.

In order to use circular plasmids as cloning vectors, the first step is to purify them. Once these cloning vectors have been purified, they are cut with a restriction nuclease (these nucleases are enzymes that cut double-stranded DNA when they recognize a specific sequence pattern) to generate linear molecules. For its part, the genomic DNA of the cell to be used in the construction of the library is cut with the same restriction nuclease; the resulting restriction fragments (including those containing the DNA to be cloned) are added to the cut plasmids and closed, forming circular recombinant DNA molecules. These recombinant molecules, which contain foreign DNA grafts, are covalently linked by the enzyme DNA ligase.

The next step in preparing the library is to introduce the circular recombinant DNA molecules into bacteria temporarily permeable to DNA; these cells are said to have been transfected with the plasmid. As these cells grow and divide - doubling in number every 30 minutes - the recombinant plasmids also replicate and produce enormous numbers of circulating DNA copies that contain foreign DNA.

Insertion of a DNA fragment into a bacterial plasmid using the ligase DNA enzyme. The plasmid is opened by a cut with a nuclease of restriction (in this case one that produces cohesive ends) and is mixed with the ligase DNA, ATP and the DNA fragment that is desired to chlorcar (which has been prepared with the same nucleas of restriction). Cohesive ends mate with each other and the ligaous DNA seals the cuts of the DNA chain, producing a complete DNA molecule

DNA ligase dysfunctions

The DNA ligase IV complex, consisting of the DNA ligase IV catalytic subunit and its cofactor XRCC4, performs the ligation step of repair. XLF, also known as Cernunnos, is homologous to yeast Nej1 and is also required for NHEJ ("No Homologue Extreme junction" is a pathway that repairs double-strand breaks in DNA. NHEJ is known as 'non-homologous' because the break ends are ligated directly without the need for a homologous template, in contrast to homologous recombination, which requires a sequence homologous to the repair leader). While the precise role of XLF is unknown, it interacts with the XRCC4/DNA ligase IV complex and is likely involved in the ligation step. Recent evidence suggests that XLF re-adenylates DNA ligase IV after ligation, reloading the ligase and allowing it to catalyze a second ligation.

DNA ligase IV and XLF are required for all NHEJ events.

Many NHEJ genes have been deleted in mice. Deletion of XRCC4 or LIG4 causes embryonic lethality in mice, indicating that NHEJ is essential for viability in mammals. All NHEJ mutant mice display a SCID phenotype, sensitivity to ionizing radiation, and neuronal apoptosis.

Partial DNA ligase dysfunction is also related to Chromosomal Instability Syndrome.