Reconstitution of the mammalian DNA double-strand break end-joining reaction reveals a requirement for an Mre11/Rad50/NBS1-containing fraction

The non-homologous end-joining pathway promotes direct enzymatic rejoining of DNA double-strand breaks (DSBs) and is an important determinant of genome stability in eukaryotic cells. Although previous work has shown that this pathway requires Ku, DNA-PKcs and the DNA ligase IV/XRCC4 complex, we found that these proteins alone did not promote efficient joining of cohesive-ended DNA fragments in a cell-free assay. To identify factors that were missing from the reaction, we screened fractions from HeLa cell extracts for the ability to stimulate the joining of cohesive DNA ends in a complementation assay containing other known proteins required for DNA DSB repair. We identified a factor that restored end-joining activity to the level observed in crude nuclear extracts. Factor activity copurified with Rad50, Mre11 and NBS1, three proteins that have previously been implicated in DSB repair by genetic and cytologic evidence. Factor activity was inhibited by anti-Mre11 antibody. The reconstituted system remained fully dependent on DNL IV/XRCC4 and at least partially dependent on Ku, but the requirement for DNA-PKcs was progressively lost as other components were purified. Results support a model where DNA-PKcs acts early in the DSB repair pathway to regulate progression of the reaction, and where Mre11, Rad50 and NBS1 play a key role in aligning DNA ends in a synaptic complex immediately prior to ligation.     

Identification of DNA-PKcs phosphorylation sites in XRCC4 and effects of mutations at these sites on DNA end joining in a cell-free system

Nonhomologous end joining (NHEJ) is the principal mechanism for repairing DNA double-strand breaks in mammalian cells. NHEJ requires at least three protein components: the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Ku protein, and the DNA ligase IV/XRCC4 (DNL IV/XRCC4) complex. Although DNA-PKcs phosphorylates several sites within itself and these other proteins, the significance of phosphorylation at individual sites is not yet understood. Here we investigate the effects of DNA-PKcs-mediated phosphorylation at two sites in XRCC4. One is a previously described site at serine 260; the other is a newly mapped site at serine 318. XRCC4 bearing mutations at these sites was co-expressed with DNL IV, the resulting complexes were purified, and activity was tested in a cell-free end-joining system reconstituted from recombinant and purified proteins. Substitution of alanine for serine 260 or 318, which prevents phosphorylation at these positions, or aspartate for serine 260, which mimics constitutive phosphorylation, had no significant effect on overall end-joining activity. In the assay system used, DNA-PKcs is not essential, but when present, arrests the reaction until phosphorylation occurs, in effect establishing a reaction checkpoint. Mutations at serines 260 and 318 did not affect establishment or release from the checkpoint. Results demonstrate that DNA-PKcs-mediated phosphorylation of XRCC4 serine 260 and serine 318 does not directly control end-joining under the conditions tested.     

Association of DNA polymerase mu (pol mu) with Ku and ligase IV: role for pol mu in end-joining double-strand break repair

Mammalian DNA polymerase mu (pol mu) is related to terminal deoxynucleotidyl transferase, but its biological role is not yet clear. We show here that after exposure of cells to ionizing radiation (IR), levels of pol mu protein increase. pol mu also forms discrete nuclear foci after IR, and these foci are largely coincident with IR-induced foci of gammaH2AX, a previously characterized marker of sites of DNA double-strand breaks. pol mu is thus part of the cellular response to DNA double-strand breaks. pol mu also associates in cell extracts with the nonhomologous end-joining repair factor Ku and requires both Ku and another end-joining factor, XRCC4-ligase IV, to form a stable complex on DNA in vitro. pol mu in turn facilitates both stable recruitment of XRCC4-ligase IV to Ku-bound DNA and ligase IV-dependent end joining. In contrast, the related mammalian DNA polymerase beta does not form a complex with Ku and XRCC4-ligase IV and is less effective than pol mu in facilitating joining mediated by these factors. Our data thus support an important role for pol mu in the end-joining pathway for repair of double-strand breaks.     

Ku recruits the XRCC4-ligase IV complex to DNA ends

Genetic experiments have determined that Ku, XRCC4, and ligase IV are required for repair of double-strand breaks by the end-joining pathway. The last two factors form a tight complex in cells. However, ligase IV is only one of three known mammalian ligases and is intrinsically the least active in intermolecular ligation; thus, the biochemical basis for requiring this ligase has been unclear. We demonstrate here a direct physical interaction between the XRCC4-ligase IV complex and Ku. This interaction is stimulated once Ku binds to DNA ends. Since XRCC4-ligase IV alone has very low DNA binding activity, Ku is required for effective recruitment of this ligase to DNA ends. We further show that this recruitment is critical for efficient end-joining activity in vitro. Preformation of a complex containing Ku and XRCC4-ligase IV increases the initial ligation rate 20-fold, indicating that recruitment of the ligase is an important limiting step in intermolecular ligation. Recruitment by Ku also allows XRCC4-ligase IV to use Ku's high affinity for DNA ends to rapidly locate and ligate ends in an excess of unbroken DNA, a necessity for end joining in cells. These properties are conferred only on ligase IV, because Ku does not similarly interact with the other mammalian ligases. We have therefore defined cell-free conditions that reflect the genetic requirement for ligase IV in cellular end joining and consequently can explain in molecular terms why this factor is required.     

Involvement of poly(ADP-ribose) polymerase-1 and XRCC1/DNA ligase III in an alternative route for DNA double-strand breaks rejoining

The efficient repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genomic integrity. In mammalian cells, the nonhomologous end-joining process that represents the predominant repair pathway relies on the DNA-dependent protein kinase (DNA-PK) and the XRCC4-DNA ligase IV complex. Nonetheless, several in vitro and in vivo results indicate that mammalian cells use more than a single end-joining mechanism. While searching for a DNA-PK-independent end-joining activity, we found that the pretreatment of DNA-PK-proficient and -deficient rodent cells with an inhibitor of the poly(ADP-ribose) polymerase-1 enzyme (PARP-1) led to increased cytotoxicity of the highly efficient DNA double-strand breaking compound calicheamicin gamma1. In addition, the repair kinetics of the DSBs induced by calicheamicin gamma1 was delayed both in PARP-1-proficient cells pretreated with the PARP-1 inhibitor and in PARP-1-deficient cells. In order to get new insights into the mechanism of an alternative route for DSBs repair, we have established a new synapsis and end-joining two-step assay in vitro, operating on DSBs with either nuclear protein extracts or recombinant proteins. We found an end-joining activity independent of the DNA-PK/XRCC4-ligase IV complex but that actually required a novel synapsis activity of PARP-1 and the ligation activity of the XRCC1-DNA ligase III complex, proteins otherwise involved in the base excision repair pathway. Taken together, these results strongly suggest that a PARP-1-dependent DSBs end-joining activity may exist in mammalian cells. We propose that this mechanism could act as an alternative route of DSBs repair that complements the DNA-PK/XRCC4/ligase IV-dependent nonhomologous end-joining.     

PARP-3 and APLF function together to accelerate nonhomologous end-joining

PARP-3 is a member of the ADP-ribosyl transferase superfamily of unknown function. We show that PARP-3 is stimulated by DNA double-strand breaks (DSBs) in vitro and functions in the same pathway as the poly (ADP-ribose)-binding protein APLF to accelerate chromosomal DNA DSB repair. We implicate PARP-3 in the accumulation of APLF at DSBs and demonstrate that APLF promotes the retention of XRCC4/DNA ligase IV complex in chromatin, suggesting that PARP-3 and APLF accelerate DNA ligation during nonhomologous end-joining (NHEJ). Consistent with this, we show that class switch recombination in Aplf(-/-) B cells is biased toward microhomology-mediated end-joining, a pathway that operates in the absence of XRCC4/DNA ligase IV, and that the requirement for PARP-3 and APLF for NHEJ is circumvented by overexpression of XRCC4/DNA ligase IV. These data identify molecular roles for PARP-3 and APLF in chromosomal DNA double-strand break repair reactions.     

The DNA-dependent protein kinase interacts with DNA to form a protein-DNA complex that is disrupted by phosphorylation

DNA double-strand breaks are a serious threat to genome stability and cell viability. One of the major pathways for the repair of DNA double-strand breaks in human cells is nonhomologous end-joining. Biochemical and genetic studies have shown that the DNA-dependent protein kinase (DNA-PK), XRCC4, DNA ligase IV, and Artemis are essential components of the nonhomologous end-joining pathway. DNA-PK is composed of a large catalytic subunit, DNA-PKcs, and a heterodimer of Ku70 and Ku80 subunits. Current models predict that the Ku heterodimer binds to ends of double-stranded DNA, then recruits DNA-PKcs to form the active protein kinase complex. XRCC4 and DNA ligase IV are subsequently required for ligation of the DNA ends. Magnesium-ATP and the protein kinase activity of DNA-PKcs are essential for DNA double-strand break repair. However, little is known about the physiological targets of DNA-PK. We have previously shown that DNA-PKcs and Ku undergo autophosphorylation, and that this correlates with loss of protein kinase activity. Here we show, using electron spectroscopic imaging, that DNA-PKcs and Ku interact with multiple DNA molecules to form large protein-DNA complexes that converge at the base of multiple DNA loops. The number of large protein complexes and the amount of DNA associated with them were dramatically reduced under conditions that promote phosphorylation of DNA-PK. Moreover, treatment of autophosphorylated DNA-PK with the protein phosphatase 1 catalytic subunit restored complex formation. We propose that autophosphorylation of DNA-PK plays an important regulatory role in DNA double-strand break repair by regulating the assembly and disassembly of the DNA-PK-DNA complex.     

APLF (C2orf13) facilitates nonhomologous end-joining and undergoes ATM-dependent hyperphosphorylation following ionizing radiation

Nonhomologous end-joining (NHEJ) is the major mammalian DNA double-strand break (DSB) repair pathway of DSBs induced by DNA damaging agents. NHEJ is initiated by the recognition of DSBs by the DNA end-binding heterodimer, Ku, and the final step of DNA end-joining is accomplished by the XRCC4-DNA ligase IV complex. We demonstrate that Aprataxin and PNK-like factor (APLF), an endo/exonuclease with an FHA domain and unique zinc fingers (ZFs), interacts with both Ku and XRCC4-DNA ligase IV in human cells. The interaction of APLF with XRCC4-DNA ligase IV is FHA- and phospho-dependent, and is mediated by CK2 phosphorylation of XRCC4 in vitro. In contrast, APLF associates with Ku independently of the FHA and ZF domains, and APLF complexes with Ku at DNA ends. APLF undergoes ionizing radiation (IR) induced ATM-dependent hyperphosphorylation at serine residue 116, which is highly conserved across mammalian APLF homologues. We demonstrate further that depletion of APLF in human cells by siRNA is associated with impaired NHEJ. Collectively, these results suggest that APLF is an ATM target that is involved in NHEJ and facilitates DSB repair, likely via interactions with Ku and XRCC4-DNA ligase IV.     

Coordinated assembly of Ku and p460 subunits of the DNA-dependent protein kinase on DNA ends is necessary for XRCC4-ligase IV recruitment

Repair of DNA double-strand breaks by the non-homologous end-joining pathway (NHEJ) requires a minimal set of proteins including DNA-dependent protein kinase (DNA-PK), DNA-ligase IV and XRCC4 proteins. DNA-PK comprises Ku70/Ku80 heterodimer and the kinase subunit DNA-PKcs (p460). Here, by monitoring protein assembly from human nuclear cell extracts on DNA ends in vitro, we report that recruitment to DNA ends of the XRCC4-ligase IV complex responsible for the key ligation step is strictly dependent on the assembly of both the Ku and p460 components of DNA-PK to these ends. Based on co-immunoprecipitation experiments, we conclude that interactions of Ku and p460 with components of the XRCC4-ligase IV complex are mainly DNA-dependent. In addition, under p460 kinase permissive conditions, XRCC4 is detected at DNA ends in a phosphorylated form. This phosphorylation is DNA-PK-dependent. However, phosphorylation is dispensable for XRCC4-ligase IV loading to DNA ends since stable DNA-PK/XRCC4-ligase IV/DNA complexes are recovered in the presence of the kinase inhibitor wortmannin. These findings extend the current knowledge of the assembly of NHEJ repair proteins on DNA termini and substantiate the hypothesis of a scaffolding role of DNA-PK towards other components of the NHEJ DNA repair process.     

XLF interacts with the XRCC4-DNA ligase IV complex to promote DNA nonhomologous end-joining

DNA nonhomologous end-joining (NHEJ) is a predominant pathway of DNA double-strand break repair in mammalian cells, and defects in it cause radiosensitivity at the cellular and whole-organism levels. Central to NHEJ is the protein complex containing DNA Ligase IV and XRCC4. By searching for additional XRCC4-interacting factors, we identified a previously uncharacterized 33 kDa protein, XRCC4-like factor (XLF, also named Cernunnos), that has weak sequence homology with XRCC4 and is predicted to display structural similarity to XRCC4. We show that XLF directly interacts with the XRCC4-Ligase IV complex in vitro and in vivo and that siRNA-mediated downregulation of XLF in human cell lines leads to radiosensitivity and impaired NHEJ. Furthermore, we establish that NHEJ-deficient 2BN cells derived from a radiosensitive and immune-deficient patient lack XLF due to an inactivating frameshift mutation in its gene, and that reintroduction of wild-type XLF into such cells corrects their radiosensitivity and NHEJ defects. XLF thus constitutes a novel core component of the mammalian NHEJ apparatus.     

Identification of human autoantibodies to the DNA ligase IV/XRCC4 complex and mapping of an autoimmune epitope to a potential regulatory region

The nonhomologous end-joining pathway is the principal mechanism for repair of ionizing radiation-induced, double-strand breaks in mammalian cells. Three polypeptides in this pathway, including the two subunits of Ku protein and the catalytic subunit of the DNA-dependent protein kinase, are known targets of autoantibodies in systemic rheumatic diseases. Here we show that two additional polypeptides in the pathway, DNA ligase IV and XRCC4, are also targets of autoantibodies. These Abs were present in 20% of patients with systemic lupus erythematosus and overlap syndrome. Previous work has shown that XRCC4 is subject to radiation-induced post-translational modification, including phosphorylation by DNA-dependent protein kinase and cleavage by caspase 3. We mapped a major autoimmune epitope in XRCC4 and found that it encompassed a DNA-dependent protein kinase phosphorylation site, which is located at serine 260; that it was adjacent to a site for caspase 3, which cleaves after residue 265; and that it also spanned a site for the inflammatory protease, granzyme B, which cleaves after residue 254. The finding that five different polypeptides in the nonhomologous end-joining pathway are potential targets of autoantibodies together with the observation that one of the autoimmune epitopes in XRCC4 coincides with a sequence that is a nexus for radiation-induced regulatory events suggest that exposure to agents that introduce DNA double-strand breaks may be one of the factors that influences the development of an autoimmune response in susceptible individuals.     

Defining interactions between DNA-PK and ligase IV/XRCC4

Non-homologous end joining (NHEJ) is a major pathway for the repair of DNA double-strand breaks (DSBs) in mammalian cells. DNA-dependent protein kinase (DNA-PK), ligase IV, and XRCC4 are all critical components of the NHEJ repair pathway. DNA-PK is composed of a heterodimeric DNA-binding component, Ku, and a large catalytic subunit, DNA-PKcs. Ligase IV and XRCC4 associate to form a multimeric complex that is also essential for NHEJ. DNA-PK and ligase IV/XRCC4 interact at DNA termini which results in stimulated ligase activity. Here, we define interactions between the components of these two essential complexes, DNA-PK and ligase IV/XRCC4. We find that ligase IV/XRCC4 associates with DNA-PK in a DNA-independent manner. The specific protein-protein interactions that mediate the interaction between these two complexes are further identified. Direct interactions between ligase IV and Ku as well as between XRCC4 and DNA-PKcs are shown. In contrast, binding of ligase IV to DNA-PKcs or XRCC4 to Ku is very weak or non-existent. Our data defines the specific protein pairs involved in the association of DNA-PK and ligase IV/XRCC4, and suggests a molecular mechanism for coordinating the assembly of the DNA repair complex at DNA breaks.     

Monoubiquitination of the nonhomologous end joining protein XRCC4

Nonhomologous end joining is one of the major pathways by which cells repair double-strand breaks, and the XRCC4-DNA ligase IV complex is required for the ligation step. To better understand the regulation and stability of XRCC4 and DNA ligase IV, we investigated the ubiquitination status of these two proteins. We identified a predominantly monoubiquitinated form of XRCC4, and higher molecular weight forms of ubiquitinated XRCC4 were detected in lower abundance. In response to etoposide-induced DNA damage, ubiquitinated XRCC4 became more pronounced and was additionally phosphorylated. We confirmed that DNA ligase IV is unstable in the absence of XRCC4, with a half-life of approximately 30-90 min. Unlike XRCC4, we did not detect ubiquitinated forms of DNA ligase IV, and we found that the presence of XRCC4 stabilized DNA ligase IV more significantly than proteasome inhibitors. Monoubiquitination of XRCC4 may play a critical role in the regulation of nonhomologous end joining.     

Live cell imaging of XLF and XRCC4 reveals a novel view of protein assembly in the non-homologous end-joining pathway

XLF, also known as Cernunnos, is a newly identified core factor of the non-homologous end-joining (NHEJ) pathway for DNA double-strand breaks (DSBs) repair. XLF is known to stimulate DNA ligase IV in vitro through its interaction with XRCC4. Here, we outline the key findings on the dynamic behavior of XLF and XRCC4 at DSBs in living cells. XLF is quickly recruited to DSBs in the absence of XRCC4 or DNA-PKcs. The recruited XLF molecules constantly exchange at DSBs, and XRCC4 modulates the exchange rate of the recruited XLF. XRCC4 can be recruited to DSBs without DNA-PKcs, but DNA-PKcs stabilizes the recruited XRCC4. These observations are inconsistent with the prevailing concept that NHEJ proteins are sequentially recruited to DSBs, which is mainly supported by in vitro evidence. We propose a novel two-phase model for the assembly of NHEJ factors to DSBs in vivo. XLF, XRCC4, and DNA-PKcs are independently recruited to Ku-bound DSBs. The recruited factors are assembled into a large complex, in which the protein interactions observed in vitro define the stability of the recruited factors. This new view has broad implications for the mechanism of DSB sensing and functional protein assembly in the NHEJ pathway.     

Binding of inositol phosphate to DNA-PK and stimulation of double-strand break repair

In mammalian cells, double-strand breaks in DNA can be repaired by nonhomologous end-joining (NHEJ), a process dependent upon Ku70/80, DNA-PKcs, XRCC4, and DNA ligase IV. Starting with HeLa cell-free extracts, which promote NHEJ in a reaction dependent upon all of these proteins, we have purified a novel factor that stimulates DNA end-joining in vitro. Using a combination of phosphorus NMR, mass spectroscopy, and strong anion exchange chromatography, we identify this factor as inositol hexakisphosphate (IP6). Purified IP6 is bound by DNA-PK and specifically stimulates DNA-PK-dependent end-joining in vitro. The involvement of inositol phosphate in DNA-PK-dependent NHEJ is of particular interest since the catalytic domain of DNA-PKcs is similar to that found in the phosphatidylinositol 3 (PI 3)-kinase family.     

Length-dependent binding of human XLF to DNA and stimulation of XRCC4.DNA ligase IV activity

An XRCC4-like factor, called XLF or Cernunnos, was recently identified as another important factor in the non-homologous DNA end joining (NHEJ) process. NHEJ is the major pathway for the repair of double-strand DNA breaks. The similarity in the putative secondary structures of XLF and XRCC4 as well as the association of XLF with XRCC4.DNA ligase IV in vivo suggested a role in the final ligation step of NHEJ. Here, we find that purified XLF directly interacts with purified XRCC4.DNA ligase IV complex and stimulates the ligase complex in a direct assay for ligation activity. Purified XLF has DNA binding activity, but this binding is dependent on DNA length in a manner most consistent with orientation of the C-terminal alpha helices parallel to the DNA helix. To better understand the function of XLF, we purified an XLF mutant (R57G), which was identified in patients with NHEJ deficiency and severe combined immunodeficiency. Surprisingly, the mutant protein retained its ability to stimulate XRCC4.DNA ligase IV but failed to translocate to the nucleus, and this appears to be the basis for the NHEJ defect in this patient.     

XRCC4:DNA ligase IV can ligate incompatible DNA ends and can ligate across gaps

XRCC4 and DNA ligase IV form a complex that is essential for the repair of all double-strand DNA breaks by the nonhomologous DNA end joining pathway in eukaryotes. We find here that human XRCC4:DNA ligase IV can ligate two double-strand DNA ends that have fully incompatible short 3' overhang configurations with no potential for base pairing. Moreover, at DNA ends that share 1-4 annealed base pairs, XRCC4:DNA ligase IV can ligate across gaps of 1 nt. Ku can stimulate the joining, but is not essential when there is some terminal annealing. Polymerase mu can add nucleotides in a template-independent manner under physiological conditions; and the subset of ends that thereby gain some terminal microhomology can then be ligated. Hence, annealing at sites of microhomology is very important, but the flexibility of the ligase complex is paramount in nonhomologous DNA end joining. These observations provide an explanation for several in vivo observations that were difficult to understand previously.     

Loss of DNA ligase IV prevents recognition of DNA by double-strand break repair proteins XRCC4 and XLF

The repair of DNA double-strand breaks by nonhomologous end-joining (NHEJ) is essential for maintenance of genomic integrity and cell viability. Central to the molecular mechanism of NHEJ is DNA ligase IV/XRCC4/XLF complex, which rejoins the DNA. During adenovirus (Ad5) infection, ligase IV is targeted for degradation in a process that requires expression of the viral E1B 55k and E4 34k proteins while XRCC4 and XLF protein levels remain unchanged. We show that in Ad5-infected cells, loss of ligase IV is accompanied by loss of DNA binding by XRCC4. Expression of E1B 55k and E4 34k was sufficient to cause loss of ligase IV and loss of XRCC4 DNA binding. Using ligase IV mutant human cell lines, we determined that the absence of ligase IV, and not expression of viral proteins, coincided with inhibition of DNA binding by XRCC4. In ligase IV mutant human cell lines, DNA binding by XLF was also inhibited. Expression of both wild-type and adenylation-mutant ligase IV in ligase IV-deficient cells restored DNA binding by XRCC4. These data suggest that the intrinsic DNA-binding activities of XRCC4 and XLF may be subject to regulation and are down regulated in human cells that lack ligase IV.     

C-terminal region of DNA ligase IV drives XRCC4/DNA ligase IV complex to chromatin

DNA ligase IV (LIG4) and XRCC4 form a complex to ligate two DNA ends at the final step of DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ). It is not fully understood how these proteins are recruited to DSBs. We recently demonstrated radiation-induced chromatin binding of XRCC4 by biochemical fractionation using detergent Nonidet P-40. In the present study, we examined the role of LIG4 in the recruitment of XRCC4/LIG4 complex to chromatin. The chromatin binding of XRCC4 was dependent on the presence of LIG4. The mutations in two BRCT domains (W725R and W893R, respectively) of LIG4 reduced the chromatin binding of LIG4 and XRCC4. The C-terminal fragment of LIG4 (LIG4-CT) without N-terminal catalytic domains could bind to chromatin with XRCC4. LIG4-CT with W725R or W893R mutation could bind to chromatin but could not support the chromatin binding of XRCC4. The ability of C-terminal region of LIG4 to interact with chromatin might provide us with an insight into the mechanisms of DSB repair through NHEJ.     

DNA binding of Xrcc4 protein is associated with V(D)J recombination but not with stimulation of DNA ligase IV activity

Mammalian cells are protected from the effects of DNA double-strand breaks by end-joining repair. Cells lacking the Xrcc4 protein are hypersensitive to agents that induce DNA double-strand breaks, and are unable to complete V(D)J recombination. The residual repair of broken DNA ends in XRCC4-deficient cells requires short sequence homologies, thus possibly implicating Xrcc4 in end alignment. We show that Xrcc4 binds DNA, and prefers DNA with nicks or broken ends. Xrcc4 also binds to DNA ligase IV and enhances its joining activity. This stimulatory effect is shown to occur at the adenylation of the enzyme. DNA binding of Xrcc4 is correlated with its complementation of the V(D)J recombination defects in XRCC4-deficient cells, but is not required for stimulation of DNA ligase IV. Thus, the ability of Xrcc4 to bind to DNA suggests functions independent of DNA ligase IV.