LIG1

DNA ligase 1 is an is_associated_with::enzyme that in humans is encoded by the LIG1 is_associated_with::gene. is_associated_with::DNA ligases are important tools for DNA replication and repair in living organisms. There are two families of DNA ligases, ATP-dependent DNA ligases and NAD+ dependent DNA ligases. Dependence upon ATP or NAD+ is conferred in the ligase-is_associated_with::adenylate formation and which substrate is to be used. ATP dependent ligases are found in is_associated_with::eukaryotes, while NAD+ dependent ligases are found in is_associated_with::prokaryotes. DNA ligase I is found in eukaryotes and therefore is in the family of ATP-dependent DNA ligases.

Discovery
Previously it had been known that DNA replication occurred through the breakage of the double DNA strand, but the mechanism of action and enzyme responsible for ligating the strands back together was unknown. In the 1960s Lehman laboratories investigated this mystery, discovering DNA ligase and it’s mechanism of action in 1967. The Gellert, Richardson, and Hurwitz laboratories are also credited for their help in the discovery of DNA ligases in the 1960s. Of the known eukaryotic DNA ligases, DNA ligase I is the only ligase involved in DNA replication making it the most studied of the ligases.



Recruitment and regulation
The LIG1 gene encodes a, 120kDa enzyme, 919 residues long, known as DNA ligase I. The DNA ligase I polypeptide contains an is_associated_with::N-terminal replication factory-targeting sequence (RFTS), followed by a is_associated_with::nuclear localization sequence (NLS), and three functional domains. The three domains consist of an N-terminal is_associated_with::DNA binding domain (DBD), and catalytic is_associated_with::nucleotidyltransferase (NTase), and is_associated_with::C-terminal is_associated_with::oligonucleotide / is_associated_with::oligosaccharide binding (OB) domains. Although the N-terminus of the peptide has no catalytic activity it is needed for activity within the cells. The N-terminus of the protein contains a replication factory-targeting sequence that is used to recruit it to sites of DNA replication known as replication factories.

Activation and recruitment of DNA Ligase I seem to be associated with posttranslational modifications. N-terminal domain is completed through is_associated_with::phosphorylation of four is_associated_with::serine residues on this domain, Ser51, Ser76, and Ser91 by is_associated_with::cyclin-dependent kinase (CDK) and Ser66 by casein kinase II (CKII). Phosphorylation of these residues (Ser66 in particular) has been shown to possibly regulate the interaction between the RFTS to the is_associated_with::proliferating cell nuclear antigen (PCNA) when Ligase I is recruited to the replication factories during is_associated_with::S-phase. Rossi et al. proposed that when Ser66 is dephosphorylated, the RFTS of Ligase I interact with PCNA, which was confirmed in vitro by Tom et al. Both data sets provide plausible evidence the N-terminal region of Ligase I plays a regulatory role in the enzymes in vivo function in the nucleus. Moreover, the identification of a cyclin binding (Cy) motif in the catalytic C-terminus domain was shown by mutational analysis to play a role in the phosphorylation of serines 91 and 76. Together, the N-terminal serines are substrates of the CDK and CKII, which appear to play an important regulatory role DNA ligase I recruitment to the replication factory during S-phase of the is_associated_with::cell cycle.



Function and mechanism
LIG1 encodes DNA ligase I, which functions in is_associated_with::DNA replication and the is_associated_with::base excision repair process.

Eukaryotic DNA ligase 1 catalyzes a reaction that is chemically universal to all ligases. DNA ligase 1 utilizes is_associated_with::adenosine triphosphate (ATP) to catalyze the energetically favorable ligation events in both is_associated_with::DNA replication and repair. During the is_associated_with::synthesis phase (S-phase) of the eukaryotic is_associated_with::cell cycle, DNA replication occurs. DNA ligase 1 is responsible for joining is_associated_with::Okazaki fragments formed during discontinuous DNA synthesis on the DNA’s lagging strand after is_associated_with::DNA polymerase δ has replaced the RNA primer nucleotides with DNA nucleotides. If the Okazaki fragments are not properly ligated together, the unligated DNA (containing a ‘nick’) could easily degrade to a is_associated_with::double strand break, a phenomenon known to cause genetic mutations. In order to ligate these fragments together, the ligase progresses through three steps:
 * 1) Addition of an is_associated_with::adenosine monophosphate (AMP) group to the enzyme,  referred to as adenylylation,
 * 2) Adenosine monophosphate transfer to the DNA and
 * 3) Nick sealing, or phosphodiester bond formation.



During is_associated_with::adenylylation, there is a is_associated_with::nucleophilic attack on the alpha phosphate of ATP from a catalytic is_associated_with::lysine resulting in the production of inorganic is_associated_with::pyrophosphate (PPi) and a covalently bound lysine-AMP intermediate in the active site of DNA ligase 1.

During the AMP transfer step, the DNA ligase becomes associated with the DNA, locates a nick and catalyzes a reaction at the 5’ phosphate site of the DNA nick. An anionic oxygen on the 5’ phosphate of the DNA nick serves as the nucleophile, attacking the alpha phosphate of the covalently bound AMP causing the AMP to be covalently bound intermediate (DNA-AMP intermediate).

In order for the phosphodiester bond to be formed, the DNA-AMP intermediate must be cleaved off. To accomplish this task, there is a nucleophilic attack on the 5’-phosphate from the upstream 3’-hydroxyl which results in the formation of the phosphodiester bond. During this nucleophilic attack, the AMP group is pushed off the 5’ phosphate as the leaving group allowing for the nick to seal and the AMP to be released, completing one cycle of DNA ligation.

Interestingly, under suboptimal conditions the ligase can disassociate from the DNA before the full reaction is complete. It has been shown that is_associated_with::magnesium levels can slow the nick sealing process, causing the ligase to disassociate from the DNA, leaving an aborted adenylylated intermediate incapable of being fixed without the aid of a is_associated_with::phosphodiesterase. is_associated_with::Aprataxin (a phosphodiesterase) has been shown to act on aborted DNA intermediates via hydrolysis of the AMP-phosphate bond, restoring the DNA to its initial state before the ligase had reacted.

Role in damaged base repair


DNA Ligase I functions to ligate single stranded DNA breaks in the final step of the is_associated_with::base excision repair (BER) pathway. The nitrogenous bases of DNA are commonly damaged by environmental hazards such as is_associated_with::reactive oxygen species, toxins, and is_associated_with::ionizing radiation. BER is the major repair pathway responsible for excising and replacing damaged bases. Ligase I is involved in the LP-BER pathway, whereas ligase III is involved in the major SN-BER pathway(2). LP-BER proceeds in 4 catalytic steps. First, a is_associated_with::DNA glycosylase cleaves the is_associated_with::N-glycosidic bond, releasing the damaged base and creating an AP site– a site that lacks a is_associated_with::purine or is_associated_with::pyrimidine base. In the next step, an AP endonuclease creates a nick at the 5' end of the AP site, generating a hanging is_associated_with::deoxyribose phosphate (dRP) residue in place of the AP site. is_associated_with::DNA polymerase then synthesizes several new bases in the 5' to 3' direction, generating a hanging stretch of DNA with the dRP at its 5' end. It is at this step that SN-BER and LP-BER diverge in mechanism – in SNBER, only a single nucleotide is added and DNA Polymerase acts as a lyase to excise the AP site. In LP-BER, several bases are synthesized, generating a hanging flap of DNA, which is cleaved by a is_associated_with::flap endonuclease. This leaves behind a nicked DNA strand that is sensed and ligated by DNA Ligase. The action of ligase I is stimulated by other LP-BER enzymes, particularly AP-endonuclease and DNA polymerase.

Clinical significance
Mutations in LIG1 that lead to DNA ligase I deficiency result in is_associated_with::immunodeficiency and increased sensitivity to DNA-damaging agents.

There is only one confirmed case of a patient exhibiting Ligase I deficiency, which resulted from an inherited mutant allele. The symptoms of this deficiency manifested as stunted growth and development and an immunodeficiency. A mouse model was made based on cell lines derived from the patient, confirming that the mutant ligase confers replication errors leading to genomic instability. Notably the mutant mice also showed increases in is_associated_with::tumorigenesis.

Ligase I has also been found to be upregulated in proliferating tumor cells, as opposed to benign tumor cell lines and normal human cells. Furthermore, it has been shown that inhibiting Ligase I expression in these cells can have a cytotoxic effect, suggesting that Ligase I inhibitors may be viable chemotherapeutic agents.

Deficiencies in is_associated_with::aprataxin, a is_associated_with::phosphodiesterase responsible for reconditioning the DNA (after DNA ligase I aborts the adenylylated DNA intermediate), has been linked to is_associated_with::neurodegeneration. This suggests that DNA is incapable of reentering the repair pathway without additional back-up machinery to correct for Ligase errors.

With the structure of DNA being well known and many of the components necessary for its manipulation, repair, and usage becoming identified and characterized, researchers are beginning to look into the development of nanoscopic machinery that would be incorporated into a living organism that would possess the ability to treat diseases, fight cancer, and release medications based on a biological stimulus provided by the organism to the nanosocpic machinery. DNA ligase would most likely have to be incorporated into such a machine.