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DNA Ligase I


DNA ligases were first discovered in bacteria and are a common trait of DNA-based life forms and have almost ubiquitous involvement in DNA transactions. They are nucleotidyltransferases (NTases) that use a high-energy cofactor, either NAD or ATP, to join single-strand breaks in double-stranded DNA by catalyzing phosphodiester bond formation in a three-step reaction mechanism.1), 2) DNA ligases work together with DNA polymerases during DNA replication and DNA repair.3) There are five biochemically distinct DNA ligase activities that have been purified from extracts of mammalian tissues.4)

DNA strand-breaks occur as a result of normal DNA transactions in undamaged cells.5) Interruptions of the phosphodiester backbone of one or both strands of duplex DNA can happen as a result of the direct action of DNA damaging agents. DNA strand breaks are generated as reaction intermediates in DNA replication, DNA excision repair and recombination. DNA ligase seals these breaks, a critical event for maintaining genomic integrity, through three steps of ligation.6) DNA ligase I is also part of a family of proteins that bind to proliferating cell nuclear antigen (PCNA) to participate in DNA replication and excision repair.


DNA ligase I is a large enzyme of 125-kDa. It is the major DNA ligase activity in proliferating mammalian cells and functions in DNA replication and DNA repair.7), 8) DNA ligase I is necessary for the efficient joining of Okazaki fragments when DNA replication is reconstituted from highly purified components.9)

All eukaryotic DNA ligases use ATP as a cofactor in the DNA joining reaction.10) During the first of three chemical steps of ligation, a phosphoamide bond (P-N) forms between the α-amino group of an active site lysine and the 5’ phosphate of AMP. In the second step of the reaction, the 5’ phosphate group of NMP is transferred from the active site lysine to a phosphorylated DNA 5’ end, forming a pyrophosphate linkage (5’P-5’P). The 5’ AMP activates the 5’ phosphate of a DNA substrate for phosphodiester bond formation. During step 3, the 3’ hydroxyl of an adjacent DNA strand attacks the 5’ phosphorylated DNA end to shift AMP and covalently join the DNA strands (Fig. 1).11), 12) Ligases can also react with DNA ends that are unsuitable for ligation. This produces a chemically stable AMP adduct, which must be removed enzymatically from the DNA to allow additional repair of the “dirty breaks.”13)

Figure 1: Enzymatic ligation of DNA. Step 1: Transfer of AMP to an active-site lysine then to the 5'-PO4 end of DNA (step 2). Step 3: the 3'-OH end of a second DNA strand attacks the 5'-PO4 to release AMP and generate the ligated DNA product.

The Chlorella virus is one of the smallest eukaryotic ligases, made up of two domains (Fig. 2). The larger N-terminal NTase domain binds ATP and has many of the active-site residues. The AMP moiety bind in an active-site pocket with the adenine ring remaining buried throughout the three-step ligation reaction as the AMP is transferred to lysine, the DNA 5’ phosphate, and eventually to water. The C-terminal oligonucleotide/oligosaccharide binding (OB)-fold domain of the Chlorella ligase assists in the step 1 transfer of AMP to the active-site lysine of the NTase domain and then works as a DNA-binding domain during steps 2 and 3. The OB-fold domain of DNA ligases binds to double-stranded DNA in the minor groove, mainly interacting with the DNA strand adjacent to the 5’ phosphate end of the nick.14)

Figure 2: Comparative anatomy of DNA ligases. Shown here are examples of viral, bacterial, and mammalian DNA ligases bound to DNA. The DNA ligases from Chlorella virus and human ligase I have similar ring-shaped structures in complex with a nicked DNA substrate. The conserved catalytic core consists of the NTase domain (green) and OB-fold domain (yellow). It contacts the 3'-OH and 5'-PO4 ends of DNA during steps 2 and 3 of the end-joining reaction. The other domain (red) in each enzyme interacts with DNA, completing the ring-shaped structure.

During step 1, residues of one of the six conserved motifs of NTases, motif VI, reside within the OB-fold domain and face the active site, where they are necessary for AMP transfer to the active-site lysine. For steps 2 and 3, the OB-fold domain turns to adjust DNA-binding residues for interactions with the DNA substrate, and the motif VI residues are exposed on the protein surface away from the active site (Fig. 3).15), 16) The deoxyribose moiety of AMP swings between the active-site lysine and the 5’ DNA phosphate during the transition from step 1 to step 2, whereas the adenine base of AMP remains fixed in the binding pocket of the NTase domain. A rotation around the glycosylic bond between adenine and 2’ deoxyribose realigns the AMP phosphate for bonding with the DNA 5’ phosphate. 17)

Figure 3: Active conformations during the step 1 reaction. The Chlorella virus mRNA-capping enzyme has a nucleotide-binding domain (green) and an OB-fold domain (yellow) like those found in DNA ligases. The inactive conformation (left side) holds step 1 residues (motif VI, pink) away from the active site. The active conformation (right side) allows the OB-fold domain to present motif VI residues to the active site of the nucleotide-binding site. Motif VI residues that are conserved among nucleotidyl transferases bind the α and β phosphates of GTP (blue) in order to position the α-phosphate for nucleophilic attack by the active site lysine.

Divalent metals are necessary for normal enzymatic activity during all three steps of end joining. The metals orient the phosphate atoms of AMP and the DNA and also neutralize the charge during phosphoryl transfer reactions. However, the number of metals and their locations during each step of end joining is still uncertain.18)

The NTase and OB-fold domains together contain a minimal catalytic unit that is conserved in all known DNA ligases. These domains harbor the active site as well as many DNA-binding residues. The catalytic core of mammalian DNA ligases is embellished with additional N- and C-terminal domains (Fig. 2), which are essential for biological activity and participate in protein-protein and/or DNA-binding interactions. For example, the three mammalian DNA ligases all require an additional N-terminal DNA binding domain (DBD) for efficient ligation activity.19)

The DBD interacts in the minor groove, contacting the DNA upstream and downstream of the nick. The DBD stabilizes the DNA in an underwound conformation with a widened minor groove that exposes the ligatable ends of the DNA to conserved residues that are important for catalytic activity. The DBD also makes protein-protein interactions with the NTase domain and the OB-fold domain, finishing a ring-shaped protein structure that encircles the DNA.20)

DNA Ligase I and PCNA

DNA ligase I is part of a family of proteins that bind to proliferating cell nuclear antigen (PCNA), a sliding clamp, through a conserved 8-amino-acid motif to participate in DNA replication and excision repair.21), 22) PCNA plays a key role in the synthesis of leading and lagging strands by tethering DNA polymerases and other replication proteins to duplex DNA. The interaction with PCNA is mainly mediated by a conserved PCNA-binding motif (PIP box) at the N terminus of DNA ligase I. This region is required for the targeting of DNA ligase I to replication factories. The interaction with PCNA is required for localization of DNA ligase I at the sites of DNA replication because inactivation of PCNA binding also eliminates targeting to replication factories. The PIP box of human DNA ligase I binds to the interdomain connector loop (IDCL) of PCNA. The DNA ligase I-PCNA complex produced on DNA has one molecule of DNA ligase I per PCNA trimer. This suggests that the binding of DNA ligase I to one PCNA monomer occludes the IDCL-binding sites on the other two PCNA subunits of the trimer.23)

The resemblance in size and shape of the rings created by PCNA and the catalytic fragment of DNA ligase I suggested that the PCNA ring encircling DNA may facilitate the shift of DNA ligase I from an extended conformation into the ring shape via interactions between one of the surfaces of the PCNA ring and regions within the catalytic fragment of DNA ligase I. DNA ligase I first docks with a PCNA trimer via the interaction between the DNA ligase I PIP box and the IDCL of one of the PCNA monomers (Fig. 4). It is thought that the region adjacent to the PIP box is flexible, allowing the catalytic fragment to engage both the PCNA ring and DNA. Despite the protein-protein interaction interfaces, a large molar excess of PCNA is required for significant stimulation of DNA ligase I activity.24)

Figure 4: Model for the interaction of human DNA ligase I with DNA-linked homotrimeric PCNA. (a) DNA ligase I in elongated conformation docks onto the PCNA ring via an interaction between the N-terminal PIP box of DNA ligase I (blue) and the interdomain connector loop of a PCNA monomer. (b) The initial docking by the PIP box that is flexibly linked to the catalytic core of ligase I facilitates an inetraction between the DBD (red) and the PCNA ring. The catalytic region remain in an extended conformation at this stage. (c) Subsequent interactions with nicked DNA orchestrate the transition of the catalytic region of DNA ligase I from the extended to a closed ring conformation with each of the domains, DBD (red), adenylation (green), and OB-fold (yellow) contacting the DNA.

The PCNA clamp is loaded onto DNA in an ATP-dependent reaction catalyzed by replication factor C (RFC). RFC inhibits DNA ligase I, although pre-incubation of RFC with PCNA alleviates this inhibition, provided that the PIP box in DNA ligase I is functional. DNA ligase I may be held in an inactive complex with RFC before it is transported to a PCNA trimer located at a DNA nick that has already been processed by DNA replication and/or excision repair activities. DNA ligase I may also interact with a complex of RFC and PCNA left behind at the DNA nick after gap-filling synthesis and end processing. It is likely that ligation by DNA ligase I is the signal for unloading of the PCNA trimer, thereby recycling PCNA for other Okazaki fragments of repair events.25)

Inactivation of the PCNA-binding site of DNA ligase I had no effect on its catalytic activity. The loss of PCNA binding compromised the capability of DNA ligase I to join Okazaki fragments. The interaction between PCNA and DNA ligase I, therefore, is critical for the subnuclear targeting of the ligase and also for coordination of the molecular transactions that happen during lagging-strand synthesis.26)

1) , 4) , 6) , 7) , 9) , 10) Tomkinson, Alan E., and Zachary B. Mackey. “Structure and Function of Mammalian DNA Ligases.” ScienceDirect. 10 Oct. 1997. Web. <>.
2) , 12) , 13) , 14) , 16) , 17) , 18) , 19) , 20) , 23) , 24) , 25) Ellenberger, Tom, and Alan E. Tomkinson. “Eukaryotic DNA Ligases: Structural and Functional Insights.” Annual Review of Biochemistry. 2008. Web. <>.
3) Soderhall, Stefan, and Tomas Lindahl. “DNA Ligases of Eukaryotes.” FEBS Letters 67.1 (1976): 1-7. PubMed. Web. <http://>
5) , 15) Tomkinson, Alan E., Sangeetha Vijayakumar, John M. Pascal, and Tom Ellenberger. “DNA Ligases: Structure, Reaction Mechanism, and Function.” Chemical Reviews 106.2 (2006): 687-99. Print
8) , 11) , 22) Vijayakumar, Sangeetha, Barbara Dziegielewska, David S. Levin, Wei Song, Jinhu Yin, Austin Yang, Yoshihiro Matsumoto, Vladimir P. Bermudez, Jerard Hurwitz, and Alan E. Tomkinson. “American Society for MicrobiologyMolecular and Cellular Biology.” Phosphorylation of Human DNA Ligase I Regulates Its Interaction with Replication Factor C and Its Participation in DNA Replication and DNA Repair. 17 Feb. 2009. Web. <>
21) , 26) Levin, David S., Allison E. McKenna, Teresa A. Motycka, Yoshihiro Matsumoto, and Alan E. Tomkinson. “Interaction between PCNA and DNA Ligase I Is Critical for Joining of Okazaki Fragments and Long-patch Base-excision Repair.” Current Biology 10.15 (2000): 919-22. Print.
chem331/dna_ligase_i.txt · Last modified: 2016/06/07 09:53 (external edit)