A double-strand break (DSB) is the most lethal form of cellular DNA damage. Such breaks can be caused by exposure to certain exogenous damaging agents, like ionizing radiation. DSBs are also normal intermediates in several cellular recombination processes, including meiotic recombination, mating type switching in yeast, and antigen receptor gene rearrangement [V(D)J recombination] in vertebrates. Regardless of the source, DSBs are repaired by one of two pathways. End joining (also termed nonhomologous end joining) rejoins broken ends, removing damaged nucleotides as necessary. In contrast, homologous recombination uses an intact copy (homolog or sister chromatid) of the broken chromosome as a template for repair dependent on processive DNA synthesis. End joining is likely to be the more error-prone pathway; nevertheless, in vertebrates it is a major pathway for general repair of DSBs. It is also the only pathway used to resolve intermediates in V(D)J recombination (reviewed, e.g., in reference 16
Genetic experiments indicate that the Ku heterodimer, XRCC4, and ligase IV are required for efficient end-joining DSB repair in all eukaryotic species examined so far. Mutation of any one of these three factors in both the yeast Saccharomyces cerevisiae
) and mammalian cells (excepting neurological lineages) (9
) have roughly equivalent effects, arguing that the functions of these three factors in the end-joining pathway are interdependent (reviewed in reference 20
). Studies of S. cerevisiae
also implicate a variety of additional factors in end joining, including the MRE11-RAD50-XRS2 complex. A complex containing orthologs to MRE11 and RAD50 has been identified in vertebrates, and although it is clearly linked in some way to DSB repair, it is not yet certain that the vertebrate version of the MRE11 complex is required for end joining (reference 33
and references therein).
In mammalian cells, end joining also typically requires the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs, or XRCC7). However, an ortholog for this factor has not been found in the fully sequenced genome of S. cerevisiae
or the nearly complete genome of Caenorhabditis elegans
). Even in species with a DNA-PKcs ortholog there are circumstances where its mutation still allows much greater levels of end joining than are observed when Ku, XRCC4, or ligase IV is mutated. Mice completely deficient in DNA-PKcs can join signal end intermediates in V(D)J recombination (2
), and embryonic stem cells from such mice possess a normal level of resistance to ionizing radiation (10
). The function of DNA-PKcs in end joining therefore may be more dispensable than that of Ku, XRCC4, or ligase IV, depending on the organism, cell type, and molecular context of the ends to be joined.
Ku is perhaps the best biochemically characterized of the factors required for end joining. Ku is a heterodimer of 83- and 70-kDa subunits (encoded by the XRCC5 and XRCC6 genes, respectively) that binds to DNA ends, including forks, single-stranded overhangs, and hairpins, with high specificity and affinity (reviewed in reference 8
). Ku can juxtapose DNA ends (5
), likely explaining its ability to generally stimulate intermolecular ligation (25
Ku also recruits DNA-PKcs to DNA ends. DNA-PKcs is a 460-kDa serine/threonine protein kinase that is activated upon binding to DNA ends. Although DNA-PKcs is most efficiently recruited to and activated through formation of a complex on DNA ends with Ku, end binding and kinase activation can also occur in vitro in a Ku-independent manner (14
). Kinase activity is required for the role of DNA-PKcs in cellular end joining, but the biologically relevant substrates are not yet known (17
XRCC4 forms a tight complex with ligase IV in cells (7
) (we will subsequently refer to this complex as X4-LIV). By itself, XRCC4 binds weakly to DNA, but this property has no apparent effect on the ligation activity of the X4-LIV complex (23
). XRCC4 also interacts weakly with (19
) and is phosphorylated by DNA-PK in vitro (7
). However, interaction with DNA-PKcs is not essential for XRCC4 function, as mutant XRCC4 lacking these phosphorylation sites is capable of complementing XRCC4-deficient cells (22
) and some XRCC4-dependent repair pathways are intact in cells lacking DNA-PKcs (10
Ligase IV protein is not stable in XRCC4-deficient cells, either in mammals or yeast (4
). While interaction of XRCC4 with ligase IV results in a modest stimulation of ligase IV activity in vitro (12
), even this complex is less effective at joining DSBs than either of the other two more abundant mammalian ligases (ligase I and ligase III) (25
). Nevertheless, both genetic (9
) and biochemical (1
) experiments argue that it is the primary ligase used for end joining. Moreover, cells defective in ligase IV cannot be complemented by overexpression of the other two ligases (13
). Ligase IV (presumably with XRCC4) may thus interact with one or more other DSB repair factors in cells and consequently becomes much more effective than the other ligases at joining DNA ends.
We demonstrate here that X4-LIV directly interacts with Ku in vitro. Ku specifically recruits X4-LIV to DNA ends, and together these proteins are capable of efficient end joining under conditions where Ku and the other mammalian ligases are not. Ku, XRCC4, and ligase IV thus cooperate to form a complex that greatly facilitates intermolecular ligation, consistent with the coordinate requirement for all three components for efficient end joining in eukaryotic species from yeast to mammals.