by David Williams
DNA ligase 4 (also known as DNA ligase IV or polydeoxyribonucleotide synthase [ATP] 4) is one of four specific types of ATP-dependent DNA ligase enzymes found in mammals. This group of enzymes possesses a shared function of resolving single strand discontinuities and breaks in double stranded DNA molecules by catalyzing the formation of phosphodiester bonds. DNA ligase 4, specifically, is implicated in two major repair/recombination processes in mammalian cells in addition to its main ligase function, namely repairing double-stranded breaks (DSBs) and carrying out V(D)J recombination.
DSBs, which are induced by ionizing radiation and other radioactive agents, are some of the most dangerous forms of damage that can affect a cell 1). They result in loss or rearrangement of genomic material, events that can lead to cell death or tumor formation. V(D)J recombination is the process by which segments of immunoglobulin genes and T-cell receptors are rearranged during development of the vertebrate immune system 2). These rearrangements are crucial in contributing to the diversity and adaptability of the mammalian immune response. DNA ligase 4 accomplishes these two disparate functions through a common pathway termed non-homologous end joining (NHEJ), a process that involves correcting a double stranded break by directly splicing together the broken ends of a DNA strand. However, DNA ligase 4 does not work alone when carrying out the functions of NHEJ; it works in junction with Xrcc4, a DNA repair protein that binds to DNA ligase 4, forming a complex that enhances ligase function 3). Cells defective in either DNA ligase 4 or Xrcc4 are hypersensitive to ionizing radiation and cannot carry out V(D)J recombination. Furthermore, DNA ligase 4 or Xrcc4 deficiency in mice causes genomic instability and embryonic lethality 4).
DNA ligase 4 contains 911 amino acids, all of which have been completely sequenced 5). All four forms of mammalian ATP-dependent DNA ligase contain a similar molecular structure consisting of two domains, a catalytic domain (CD or Domain 1) and a non-catalytic domain (NCD or Domain 2) 6). These two domains are also called the adenylation domain and oligo-binding domain, respectively.
Figure 1: Important Structural Regions of DNA Ligase 4
Six conserved motifs (designated I, III, IIIa, IV, V, and VI) have been identified among DNA ligases, of which five are found in the CD. I, III, IIIA, IV, and V are essential for ATP binding and the auto-adenylation reaction involved in ligation function. Motif I, containing a critically important lysine residue, forms the active site loop of the enzyme and comprises part of the ATP binding pocket 7). In addition to these two domains, DNA ligase 4 contains an additional N-terminal DNA-binding domain (DBD) that is required for efficient ligation and enables these ligases to encircle DNA 8). Together, the CD, NCD, and DBD make up the catalytic core of DNA ligase 4. Following the N-terminal catalytic core of DNA ligase 4 is a large C-terminal region containing a tandem repeat of BRCT domains, a structural motif that is commonly found in proteins involved in DNA repair and the signaling of DNA damage 9). It is this C-terminal tail that is responsible for binding to Xrcc4, a partner protein upon which the ligase depends for stability and activity 10). This interaction occurs at a conserved binding site located within a short linker sequence of around 100 amino acids that separates the two BRCT domains 11).
The molecular structure of Xrcc4 is worth noting in order to better understand the interactions between the two proteins. Xrcc4 contains 334 amino acids and exhibits a globular N-terminal head domain that folds into a seven-stranded, trumpet-like β-barrel followed by a long helical tail 12). In vivo, the protein is in a state of dynamic equilibrium between its tetrameric form and dimeric form. Like DNA ligase 4, Xrcc4 is functionally active in V(D)J recombination; an Xrcc4 fragment spanning residues 18-204 plays a key role in the process 13).
The complex formed between DNA ligase 4 and Xrcc4 is incredibly strong; the two proteins copurify over a broad spectrum of chromatographic techniques, and their associations withstand many harsh buffer conditions 14). Key sites involved in interactions between the two proteins are amino acid residues 748-784 of DNA ligase 4 (the linker sequence between its two BRCT domains) and residues 180-213 of Xrcc4 15). Various studies have shown the stoichiometry between DNA ligase 4 and Xrcc4 within the complex to be 1:2, respectively. Analysis of the complex via x-ray crystallography shows that a solitary polypeptide chain from DNA ligase 4 interacts with the same region of both chains of one Xrcc4 dimer 16). This sequence of amino acids of DNA ligase 4 (755-782) folds into a flat, slab-like motif – comprising a β hairpin next to a short α helix – that lies across the surfaces of the side-by-side Xrcc4 monomer tails 17).
Figure 2: Interactions between Xrcc4 dimer and Ligase 4
Interactions between the Xrcc4 monomers and the DNA ligase 4 linker sequence are numerous, covering a 1,800 Å2 swath of polar and hydrophobic surface area 18). Worth mentioning is the presence of a network of charged hydrogen bonds that take place at the heart of the Xrcc4-DNA ligase 4 interface and are completely buried. There are also extensive hydrophobic contacts that govern the interface between the Xrcc4 dimer and DNA ligase 4 monomer 19).