Double-strand breaks (DSBs) in DNA pose a lethal threat to a cell: if not repaired, DSBs can cause cell death; if repaired incorrectly, DSBs can cause mutations and chromosomal rearrangements that can lead to the loss of growth control and cancer. DSBs occur naturally as a result of oxidative metabolism and V(D)J recombination in lymphocytes, as well as in response to ionising radiation and to chemotherapeutic agents such as bleomycin.
Cells have evolved a number of ways of dealing with the damage arising from DSBs: homologous recombination (HR), non-homologous end joining (NHEJ), and single-strand annealing. In mammalian cells, HR and NHEJ predominate. HR is the major repair pathway following DNA replication, using an undamaged sister chromatid as a template to accurately repair the damage. NHEJ is used to repair DSBs arising from DNA-damaging agents such as ionising radiation, and for those arising from V(D)J recombination. Unlike HR, NHEJ is an inherently error-prone process, whereby two DNA ends are joined directly without the need for sequence homology, although in some cases small regions of homology may exist in the single-stranded overhangs in the break. NHEJ can result in the loss of nucleotides from the site of breakage, which can be deleterious if the break occurs in a gene or its controlling sequences; yet this may still prove more beneficial to a cell than leaving the breaks unrepaired. There are several proteins known to be involved in NHEJ, one of which is DNA ligase IV.
Domain structure of DNA ligase IV.
The structure of the red-boxed region has been solved by crystallography.
Reprinted from Genome Biology 3(4), I. V. Martin and S. A. MacNeill, ATP-dependent DNA ligases, 2002, REVIEWS3005, PMID: 11983065.
DNA ligase IV, which is conserved in all eukaryotes, is part of a family of ATP-dependent DNA ligases that are involved in DNA replication, recombination and repair. These DNA ligases have two common domains: a catalytic domain (CD) that contains several conserved nucleotide-binding motifs, and a conserved non-catalytic domain (NCD). In addition, DNA ligase IV has a long C-terminal extension comprising of two BRCT domains (after the C-terminal domain of a breast cancer susceptibility protein, BRCA1), which are phosphopeptide-binding modules found in many proteins that regulate DNA damage responses, such as BRCA1, MDC1 and BARD1. A short linker region that is required for the binding of the XRCC4 protein, which is important for ligase activity, connects the two BRCT domains.
DNA ligase IV is a nuclear enzyme that joins the breaks in the phosphodiester backbone of DNA. The reaction mechanism involves the formation of a covalent enzyme-AMP intermediate from the cleavage of ATP to AMP and pyrophosphate. The adenylate group from AMP is then transferred to the 5’-phosphate of the nicked DNA molecule. Finally, the DNA ligase seals the gap by phosphodiester bond formation, via the displacement of the AMP residue with the 3’-hydroxyl group from the adjacent DNA strand.