Biology:LIG1

Short description: Protein-coding gene in the species Homo sapiens

Generic protein structure example

DNA ligase 1 is an enzyme that in humans is encoded by the LIG1 gene. DNA ligase I is the only known eukaryotic DNA ligase involved in both DNA replication and repair, making it the most studied of the ligases.

Discovery

It was known that DNA replication occurred through the breakage of the double DNA strand, but the enzyme responsible for ligating the strands back together, and mechanism of action, was unknown until Lehman, Gellert, Richardson, and Hurwitz laboratories, made significant contributions to the discovery of DNA ligase in 1967.

DNA Ligase I repairing nicked DNA

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 N-terminal replication factory-targeting sequence (RFTS), followed by a nuclear localization sequence (NLS), and three functional domains. The three domains consist of an N-terminal DNA binding domain (DBD), and catalytic nucleotidyltransferase (NTase), and C-terminal oligonucleotide / 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 phosphorylation of four serine residues on this domain, Ser51, Ser76, and Ser91 by 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 proliferating cell nuclear antigen (PCNA) when ligase I is recruited to the replication factories during 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 cell cycle.

DNA ligase I LIG1 gene

Function and mechanism

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

Eukaryotic DNA ligase 1 catalyzes a reaction that is chemically universal to all ligases. DNA ligase 1 utilizes adenosine triphosphate (ATP) to catalyze the energetically favorable ligation events in both DNA replication and repair. During the synthesis phase (S-phase) of the eukaryotic cell cycle, DNA replication occurs. DNA ligase 1 is responsible for joining Okazaki fragments formed during discontinuous DNA synthesis on the DNA's lagging strand after 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 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 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.

Minimalistic mechanism of DNA nick sealing by DNA ligase

During adenylylation, there is a nucleophilic attack on the alpha phosphate of ATP from a catalytic lysine resulting in the production of inorganic 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.

Under suboptimal conditions the ligase can disassociate from the DNA before the full reaction is complete. It has been shown that 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 phosphodiesterase. 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

File:BER basic pathway.svg

DNA ligase I functions to ligate single stranded DNA breaks in the final step of the base excision repair (BER) pathway. The nitrogenous bases of DNA are commonly damaged by environmental hazards such as reactive oxygen species, toxins, and 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 DNA glycosylase cleaves the N-glycosidic bond, releasing the damaged base and creating an AP site– a site that lacks a purine or pyrimidine base. In the next step, an AP endonuclease creates a nick at the 5' end of the AP site, generating a hanging deoxyribose phosphate (dRP) residue in place of the AP site. 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 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 immunodeficiency and increased sensitivity to DNA-damaging agents.

There are rare reports of patients exhibiting ligase I deficiency which resulted from inherited mutant alleles. The first case 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 tumorigenesis. Molecular, cellular, and clinical features of 5 patients from 3 kindreds with biallelic mutations were reported. The patients exhibited hypogammaglobulinemia, lymphopenia, increased proportions of circulating γδT cells, and very large red cells (macrocytosis.) Clinical severity ranged from a mild antibody deficiency to a combined immunodeficiency requiring hematopoietic stem cell transplantation. Chemical and radiation defects were demonstrated to impair the DNA repair pathways. Defects in DNA ligase 1 can thus lead to different forms of autosomal recessive, partial DNA ligase 1 deficiency leading to an immunodeficiency of variable severity.

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 aprataxin, a phosphodiesterase responsible for reconditioning the DNA (after DNA ligase I aborts the adenylylated DNA intermediate), has been linked to 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.

Further reading

• "Targeting and association of proteins with functional domains in the nucleus: the insoluble solution". Structural and Functional Organization of the Nuclear Matrix. International Review of Cytology. 162B. 1996. pp. 303–35. doi:10.1016/S0074-7696(08)62620-0. ISBN 9780123645661. 
• "Structure and function of mammalian DNA ligases". Mutation Research 407 (1): 1–9. February 1998. doi:10.1016/s0921-8777(97)00050-5. PMID 9539976. 
• "[Paralysis following Dimer X radiculography]". La Nouvelle Presse Médicale 5 (17): 1120–2. April 1976. PMID 934827. 
• "Growth retardation and immunodeficiency in a patient with mutations in the DNA ligase I gene". Lancet 339 (8808): 1508–9. June 1992. doi:10.1016/0140-6736(92)91266-B. PMID 1351188. 
• "Mutations in the DNA ligase I gene of an individual with immunodeficiencies and cellular hypersensitivity to DNA-damaging agents". Cell 69 (3): 495–503. May 1992. doi:10.1016/0092-8674(92)90450-Q. PMID 1581963. 
• "Assignment of the gene encoding DNA ligase I to human chromosome 19q13.2-13.3". Genomics 12 (1): 164–6. January 1992. doi:10.1016/0888-7543(92)90422-O. PMID 1733856. https://zenodo.org/record/1258593. 
• "A wild-type DNA ligase I gene is expressed in Bloom's syndrome cells". Proceedings of the National Academy of Sciences of the United States of America 88 (17): 7615–9. September 1991. doi:10.1073/pnas.88.17.7615. PMID 1881902. Bibcode: 1991PNAS...88.7615P. 
• "Mammalian DNA ligases. Biosynthesis and intracellular localization of DNA ligase I". The Journal of Biological Chemistry 265 (21): 12618–22. July 1990. doi:10.1016/S0021-9258(19)38388-7. PMID 2197279. 
• "Human DNA ligase I cDNA: cloning and functional expression in Saccharomyces cerevisiae". Proceedings of the National Academy of Sciences of the United States of America 87 (17): 6679–83. September 1990. doi:10.1073/pnas.87.17.6679. PMID 2204063. Bibcode: 1990PNAS...87.6679B. 
• "The N-terminal domain of human DNA ligase I contains the nuclear localization signal and directs the enzyme to sites of DNA replication". The EMBO Journal 14 (21): 5379–86. November 1995. doi:10.1002/j.1460-2075.1995.tb00222.x. PMID 7489727. 
• "Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides". Gene 138 (1–2): 171–4. January 1994. doi:10.1016/0378-1119(94)90802-8. PMID 8125298. 
• "Fluorescence in situ hybridization mapping of human chromosome 19: cytogenetic band location of 540 cosmids and 70 genes or DNA markers". Genomics 15 (1): 133–45. January 1993. doi:10.1006/geno.1993.1021. PMID 8432525. https://zenodo.org/record/1229582. 
• "Isolation and characterization of the human MRE11 homologue". Genomics 29 (1): 80–6. September 1995. doi:10.1006/geno.1995.1217. PMID 8530104. 
• "DNA ligase I is required for fetal liver erythropoiesis but is not essential for mammalian cell viability". Nature Genetics 13 (4): 489–91. August 1996. doi:10.1038/ng0896-489. PMID 8696349. 
• "Construction and characterization of a full length-enriched and a 5'-end-enriched cDNA library". Gene 200 (1–2): 149–56. October 1997. doi:10.1016/S0378-1119(97)00411-3. PMID 9373149. 
• "The replication factory targeting sequence/PCNA-binding site is required in G(1) to control the phosphorylation status of DNA ligase I". The EMBO Journal 18 (20): 5745–54. October 1999. doi:10.1093/emboj/18.20.5745. PMID 10523317. 
• "Reconstitution of proliferating cell nuclear antigen-dependent repair of apurinic/apyrimidinic sites with purified human proteins". The Journal of Biological Chemistry 274 (47): 33703–8. November 1999. doi:10.1074/jbc.274.47.33703. PMID 10559261. 
• "DNA repair patch-mediated double strand DNA break formation in human cells". The Journal of Biological Chemistry 275 (35): 27386–92. September 2000. doi:10.1074/jbc.M003126200. PMID 10827190. 

External links

• Overview of all the structural information available in the PDB for UniProt: P18858 (DNA ligase 1) at the PDBe-KB.