Detailed Introduction to Ligase

Ligase is an enzyme that catalyzes the joining of two molecules into one molecule or the end to end of a molecule. This reaction is coupled with the decomposition reaction of ATP. When the two molecules are connected, the high-energy phosphate bond of adenosine triphosphate (ATP) is broken, such as DNA ligase. Ligase is an important class in enzyme classification (EC 6): 6.1 forms C-O bonds; 6.2 forms C-S bonds; 6.3 forms C-N bonds; 6.4 forms C-C bonds. DNA ligase was first discovered at the same time by three laboratories in 1967. After decades of research by scientists, it has been discovered that DNA ligases have been found in viruses, bacteria and eukaryotes. Different types of DNA ligases have different mechanisms of action. In general, DNA ligase can be divided into adenosine triphosphate (ATP) dependent type and nicotinamide adenine dinucleotide (NAD) dependent type according to the energy source required for the reaction catalyzed by DNA ligase. Dependent DNA ligase is widely present in eukaryotes, archaea, eubacteria and viruses, while nicotinamide adenine dinucleotide dependent DNA ligase is distributed in archaea, eubacteria and viruses. Common types and mechanism of actionrn1. T4 DNA LigasernT4 DNA ligase is a DNA ligase coded by viral genomes that is currently widely used, and is widely used in gene recombination. Current studies have found that T4 phage can synthesize T4 DNA ligase, and it has been able to extract the enzyme from E. coli infected with T4 bacteriophage. In addition, scientists have also located the synthetic gene of the enzyme, the 30 gene of bacteriophage T4. T4 DNA ligase has the function of connecting sticky ends and flat ends. The researchers summarized the ligation process of DNA fragments and believe that the entire DNA ligation reaction can be completed in three consecutive steps: (1) ATP releases energy by breaking the last high-energy phosphate bond, and the simultaneously produced AMP and T4 DNA ligase use the energy released by ATP hydrolysis to form an E-AMP complex. (2) The formed E-AMP complex can recognize the nick cut before the DNA double strand. After recognition, the AMP will break away from the E-AMP complex and bind to the 5'-P group of the DNA. (3) The 3'-OH of another piece of DNA molecule that needs to be connected will attack the second step to form 5'-P-AMP, and the 3'-OH will react with the phosphate residues to form a phosphodiester bond and release an AMP at the same time. 2. Eukaryotic DNA LigasernThere are three ATP-dependent DNA ligases in eukaryotes—DNA ligase Ⅰ, DNA ligase Ⅲ and DNA ligase Ⅳ. Studies have shown that DNA ligase I and DNA ligase IV are widely distributed in eukaryotes, such as the plant kingdom and animal kingdom, while DNA ligase III is mainly distributed in vertebrates. At present, scientists believe that DNA ligase I may play a major role in the DNA replication process in eukaryotes. The regulation of DNA ligase I activity is related to its amino terminus, and there is a specific site near the active center at its amino terminus. This part can regulate the activity of DNA ligase I through phosphorylation. The function of DNA ligase I is to connect the discontinuous lagging strand (Okazaki fragment) formed at the replication fork during DNA replication. In this process, DNA Ligase I does not work alone, but needs to work closely with a variety of protein factors. During DNA replication in eukaryotes, the lagging strand is catalyzed and synthesized by DNA polymerase α. DNA polymerase α first catalyzes the synthesis of a primer, and then relies on DNA polymerase δ and proliferating cell nuclear antigen (continuous replication factor) to synthesize DNA from the primer, and then the primer is degraded. In addition, DNA ligase I also plays an important role in the base repair of damaged DNA. First, the specific endonuclease and exonuclease in the cell specifically recognize the damage site, cut near the DNA single-stranded damage site, and excise a piece of DNA. Then DNA polymerase uses another complete DNA strand as a template to repair and synthesize this piece of DNA again, and finally the gap is connected by DNA ligase I.