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.     

Interplay between Cernunnos-XLF and nonhomologous end-joining proteins at DNA ends in the cell

Cernunnos-XLF is the most recently identified core component in the nonhomologous end-joining (NHEJ) pathway for the repair of DNA double strand breaks (DSBs) in mammals. It associates with the XRCC4/ligase IV ligation complex and stimulates its activity in a still unknown manner. NHEJ also requires the DNA-dependent protein kinase that contains a Ku70/Ku80 heterodimer and the DNA-dependent protein kinase catalytic subunit. To understand the interplay between Cernunnos-XLF and the other proteins implicated in the NHEJ process, we have analyzed the interactions of Cernunnos-XLF and NHEJ proteins in cells after treatment with DNA double strand-breaking agents by means of a detergent-based cellular fractionation protocol. We report that Cernunnos-XLF is corecruited with the core NHEJ components on chromatin damaged with DSBs in human cells and is phosphorylated by the DNA-dependent protein kinase catalytic subunit. Our data show a pivotal role for DNA ligase IV in the NHEJ ligation complex assembly and recruitment to DSBs because the association of Cernunnos-XLF with the XRCC4/ligase IV complex relies primarily on the DNA ligase IV component, and an intact XRCC4/ligase IV complex is necessary for Cernunnos-XLF mobilization to damaged chromatin. Conversely, a Cernunnos-XLF defect has no apparent impact on the XRCC4/ligase IV association and recruitment to the DSBs or on the stimulation of the DNA-dependent protein kinase on DNA ends.     

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.     

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.     

Single-stranded DNA ligation and XLF-stimulated incompatible DNA end ligation by the XRCC4-DNA ligase IV complex: influence of terminal DNA sequence

The double-strand DNA break repair pathway, non-homologous DNA end joining (NHEJ), is distinctive for the flexibility of its nuclease, polymerase and ligase activities. Here we find that the joining of ends by XRCC4-ligase IV is markedly influenced by the terminal sequence, and a steric hindrance model can account for this. XLF (Cernunnos) stimulates the joining of both incompatible DNA ends and compatible DNA ends at physiologic concentrations of Mg²⁺, but only of incompatible DNA ends at higher concentrations of Mg²⁺, suggesting charge neutralization between the two DNA ends within the ligase complex. XRCC4-DNA ligase IV has the distinctive ability to ligate poly-dT single-stranded DNA and long dT overhangs in a Ku- and XLF-independent manner, but not other homopolymeric DNA. The dT preference of the ligase is interesting given the sequence bias of the NHEJ polymerase. These distinctive properties of the XRCC4-DNA ligase IV complex explain important aspects of its in vivo roles.     

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.     

Polynucleotide kinase and aprataxin-like forkhead-associated protein (PALF) acts as both a single-stranded DNA endonuclease and a single-stranded DNA 3' exonuclease and can participate in DNA end joining in a biochemical system

Polynucleotide kinase and aprataxin-like forkhead-associated protein (PALF, also called aprataxin- and PNK-like factor (APLF)) has been shown to have nuclease activity and to use its forkhead-associated domain to bind to x-ray repair complementing defective repair in Chinese hamster cells 4 (XRCC4). Because XRCC4 is a key component of the ligase IV complex that is central to the nonhomologous DNA end joining (NHEJ) pathway, this raises the possibility that PALF might play a role in NHEJ. For this reason, we further studied the nucleolytic properties of PALF, and we searched for any modulation of PALF by NHEJ components. We verified that PALF has 3' exonuclease activity. However, PALF also possesses single-stranded DNA endonuclease activity. This single-stranded DNA endonuclease activity can act at all single-stranded sites except those within four nucleotides 3' of a double-stranded DNA junction, suggesting that PALF minimally requires approximately four nucleotides of single-strandedness. Ku, DNA-dependent protein kinase catalytic subunit, and XRCC4-DNA ligase IV do not modulate PALF nuclease activity on single-stranded DNA or overhangs of duplex substrates. PALF does not open DNA hairpins. However, in a reconstituted end joining assay that includes Ku, XRCC4-DNA ligase IV, and PALF, PALF is able to resect 3' overhanging nucleotides and permit XRCC4-DNA ligase IV to complete the joining process in a manner that is as efficient as Artemis. Reduction of PALF in vivo reduces the joining of incompatible DNA ends. Hence, PALF can function in concert with other NHEJ proteins.     

Human DNA ligase IV and the ligase IV/XRCC4 complex: analysis of nick ligation fidelity

In addition to linking nicked/fragmented DNA molecules back into a contiguous duplex, DNA ligases also have the capacity to influence the accuracy of DNA repair pathways via their tolerance/intolerance of nicks containing mismatched base pairs. Although human DNA ligase I (Okazaki fragment processing) and the human DNA ligase III/XRCC1 complex (general DNA repair) have been shown to be relatively intolerant of nicks containing mismatched base pairs, the human DNA ligase IV/XRCC4 complex has not been studied in this regard. Ligase IV/XRCC4 is the sole DNA ligase involved in the repair of double strand breaks (DSBs) via the non-homologous end joining (NHEJ) pathway. During the repair of DSBs generated by chemical/physical damage as well as the repair of the programmed DSB intermediates of V(D)J recombination, there are scenarios where, at least conceptually, a capacity for ligating nicks containing mismatched base pairs would appear to be advantageous. Herein we examine whether ligase IV/XRCC4 can contribute a mismatched nick ligation activity to NHEJ. Toward this end, we (i) describe an E. coli-based coexpression system that provides relatively high yields of the ligase IV/XRCC4 complex, (ii) describe a unique rate-limiting step, which has bearing on how the complex is assayed, (iii) specifically analyze how XRCC4 influences ligase IV catalysis and substrate specificity, and (iv) probe the mismatch tolerance/intolerance of DNA ligase IV/XRCC4 via quantitative in vitro kinetic analyses. Analogous to most other DNA ligases, ligase IV/XRCC4 is shown to be fairly intolerant of nicks containing mismatched base pairs. These results are discussed in light of the biological roles of NHEJ.     

A biochemically defined system for mammalian nonhomologous DNA end joining

Nonhomologous end joining (NHEJ) is a major pathway in multicellular eukaryotes for repairing double-strand DNA breaks (DSBs). Here, the NHEJ reactions have been reconstituted in vitro by using purified Ku, DNA-PK(cs), Artemis, and XRCC4:DNA ligase IV proteins to join incompatible ends to yield diverse junctions. Purified DNA polymerase (pol) X family members (pol mu, pol lambda, and TdT, but not pol beta) contribute to junctional additions in ways that are consistent with corresponding data from genetic knockout mice. The pol lambda and pol mu contributions require their BRCT domains and are both physically and functionally dependent on Ku. This indicates a specific biochemical function for Ku in NHEJ at incompatible DNA ends. The XRCC4:DNA ligase IV complex is able to ligate one strand that has only minimal base pairing with the antiparallel strand. This important aspect of the ligation leads to an iterative strand-processing model for the steps of NHEJ.     

Conserved interactions of the splicing factor Ntr1/Spp382 with proteins involved in DNA double-strand break repair and telomere metabolism

The ligation of DNA double-strand breaks in the process of non-homologous end-joining (NHEJ) is accomplished by a heterodimeric enzyme complex consisting of DNA ligase IV and an associated non-catalytic factor. This DNA ligase also accounts for the fatal joining of unprotected telomere ends. Hence, its activity must be tightly controlled. Here, we describe interactions of the DNA ligase IV-associated proteins Lif1p and XRCC4 of yeast and human with the putatively orthologous G-patch proteins Ntr1p/Spp382p and NTR1/TFIP11 that have recently been implicated in mRNA splicing. These conserved interactions occupy the DNA ligase IV-binding sites of Lif1p and XRCC4, thus preventing the formation of an active enzyme complex. Consistently, an excess of Ntr1p in yeast reduces NHEJ efficiency in a plasmid ligation assay as well as in a chromosomal double-strand break repair (DSBR) assay. Both yeast and human NTR1 also interact with PinX1, another G-patch protein that has dual functions in the regulation of telomerase activity and telomere stability, and in RNA processing. Like PinX1, NTR1 localizes to telomeres and associates with nucleoli in yeast and human cells, suggesting a function in localized control of DSBR.     

Crystal structure of the Xrcc4 DNA repair protein and implications for end joining

XRCC4 is essential for carrying out non-homologous DNA end joining (NHEJ) in all eukaryotes and, in particular, V(D)J recombination in vertebrates. Xrcc4 protein forms a complex with DNA ligase IV that rejoins two DNA ends in the last step of V(D)J recombination and NHEJ to repair double strand breaks. XRCC4-defective cells are extremely sensitive to ionizing radiation, and disruption of the XRCC4 gene results in embryonic lethality in mice. Here we report the crystal structure of a functional fragment of Xrcc4 at 2.7 A resolution. Xrcc4 protein forms a strikingly elongated dumb-bell-like tetramer. Each of the N-terminal globular head domains consists of a beta-sandwich and a potentially DNA-binding helix- turn-helix motif. The C-terminal stalk comprising a single alpha-helix >120 A in length is partly incorporated into a four-helix bundle in the Xrcc4 tetramer and partly involved in interacting with ligase IV. The Xrcc4 structure suggests a possible mode of coupling ligase IV association with DNA binding for effective ligation of DNA ends.     

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.     

Targeted disruption of the gene encoding DNA ligase IV leads to lethality in embryonic mice

DNA ligase IV is the most recently identified member of a family of enzymes joining DNA strand breaks in mammalian cell nuclei [1] and [2]. The enzyme occurs in a complex with the XRCC4 gene product [3], an interaction mediated via its unique carboxyl terminus [4] and [5]. Cells lacking XRCC4 are hypersensitive to ionising radiation and defective in V(D)J recombination [3] and [6], implicating DNA ligase IV in the pathway of nonhomologous end-joining (NHEJ) of DNA double-strand breaks mediated by XRCC4, the Ku70/80 heterodimer and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in mammalian cells (reviewed in [7]). The phenotype of a null mutant of the Saccharomyces cerevisiae DNA ligase IV homologue indicates that the enzyme is non-essential and functions in yeast NHEJ [8], [9] and [10]. Unlike other mammalian DNA ligases for which cDNAs have been characterised, DNA ligase IV is encoded by an intronless gene (LIG4). Here, we show that targeted disruption of LIG4 in the mouse leads to lethality associated with extensive apoptotic cell death in the embryonic central nervous system. Thus, unlike Ku70/80 and DNA-PKcs [11], [12], [13] and [14], DNA ligase IV has an essential function in early mammalian development.     

Lif1p targets the DNA ligase Lig4p to sites of DNA double-strand breaks

DNA ligases catalyse the joining of DNA single- and double-strand breaks. Saccharomyces cerevisiae Cdc9p is a homologue of mammalian DNA ligase I and is required for DNA replication, recombination and single-strand break repair. The other yeast ligase, Lig4p/Dnl4p, is a homologue of mammalian DNA ligase IV, and functions in the non-homologous end-joining (NHEJ) pathway of DNA double-strand break repair [1] [2] [3] [4]. Lig4p interacts with Lif1p, the yeast homologue of the human ligase IV-associated protein, XRCC4 [5]. This interaction takes place through the carboxy-terminal domain of Lig4p and is required for Lig4p stability. We show that the carboxy-terminal interaction region of Lig4p is necessary for NHEJ but, when fused to Cdc9p, is insufficient to confer NHEJ function to Cdc9p. Also, Lif1p stimulates the in vitro catalytic activity of Lig4p in adenylation and DNA ligation. Nevertheless, Lig4p is inactive in NHEJ in the absence of Lif1p in vivo, even when Lig4p is stably expressed. We show that Lif1p binds DNA in vitro and, through in vivo cross-linking and chromatin immuno precipitation assays, demonstrate that it targets Lig4p to chromosomal DNA double-strand breaks. Furthermore, this targeting requires another key NHEJ protein, Ku.     

DNA polymerase lambda can elongate on DNA substrates mimicking non-homologous end joining and interact with XRCC4-ligase IV complex

Non-homologous end joining (NHEJ) is one of two pathways responsible for the repair of double-strand breaks in eukaryotic cells. The mechanism involves the alignment of broken DNA ends with minimal homology, fill in of short gaps by DNA polymerase(s), and ligation by XRCC4-DNA ligase IV complex. The gap-filling polymerase has not yet been positively identified, but recent biochemical studies have implicated DNA polymerase lambda (pol lambda), a novel DNA polymerase that has been assigned to the pol X family, in this process. Here we demonstrate that purified pol lambda can efficiently catalyze gap-filling synthesis on DNA substrates mimicking NHEJ. By designing two truncated forms of pol lambda, we also show that the unique proline-rich region in pol lambda plays a role in limiting strand displacement synthesis, a feature that may help its participation in in vivo NHEJ. Moreover, pol lambda interacts with XRCC4-DNA ligase IV via its N-terminal BRCT domain and the interaction stimulates the DNA synthesis activity of pol lambda. Taken together, these data strongly support that pol lambda functions in DNA polymerization events during NHEJ.     

Repair of DNA double strand breaks by non-homologous end joining

DNA double strand breaks (DSB) are the most serious form of DNA damage. If not repaired they can lead to cell death. If misrepaired DSBs contribute to chromosomal aberrations and genomic instability. Non-homologous end joining (NHEJ) is one of two major pathways for the repair of DSBs in human cells. Proteins known to be required for NHEJ include the DNA-dependent protein kinase (DNA-PK), XRCC4, DNA ligase IV, and Artemis. This review discusses how these and other accessory proteins may function in the repair of DSBs produced by ionizing radiation (IR) and by V(D)J recombination.     

Evolutionary and functional conservation of the DNA non-homologous end-joining protein, XLF/Cernunnos

Non-homologous end-joining is a major pathway of DNA double-strand break repair in mammalian cells, deficiency in which confers radiosensitivity and immune deficiency at the whole organism level. A core protein complex comprising the Ku70/80 heterodimer together with a complex between DNA ligase IV and XRCC4 is conserved throughout eukaryotes and assembles at double-strand breaks to mediate ligation of broken DNA ends. In Saccharomyces cerevisiae an additional NHEJ protein, Nej1p, physically interacts with the ligase IV complex and is required in vivo for ligation of DNA double-strand breaks. Recent studies with cells derived from radiosensitive and immune-deficient patients have identified the human protein, XLF (also named Cernunnos), as a crucial NHEJ protein. Here we show that XLF and Nej1p are members of the same protein superfamily and that this family has members in diverse eukaryotes. Indeed, we show that a member of this family encoded by a previously uncharacterized open-reading frame in the Schizosaccharomyces pombe genome is required for NHEJ in this organism. Furthermore, our data reveal that XLF family proteins can bind to DNA and directly interact with the ligase IV-XRCC4 complex to promote DSB ligation. We therefore conclude that XLF family proteins interact with the ligase IV-XRCC4 complex to constitute the evolutionarily conserved enzymatic core of the NHEJ machinery.     

Different DNA End Configurations Dictate Which NHEJ Components Are Most Important for Joining Efficiency

The nonhomologous DNA end-joining (NHEJ) pathway is a key mechanism for repairing dsDNA breaks that occur often in eukaryotic cells. In the simplest model, these breaks are first recognized by Ku, which then interacts with other NHEJ proteins to improve their affinity at DNA ends. These include DNA-PK_cs and Artemis for trimming the DNA ends; DNA polymerase μ and λ to add nucleotides; and the DNA ligase IV complex to ligate the ends with the additional factors, XRCC4 (X-ray repair cross-complementing protein 4), XLF (XRCC4-like factor/Cernunos), and PAXX (paralog of XRCC4 and XLF). In vivo studies have demonstrated the degrees of importance of these NHEJ proteins in the mechanism of repair of dsDNA breaks, but interpretations can be confounded by other cellular processes. In vitro studies with NHEJ proteins have been performed to evaluate the nucleolytic resection, polymerization, and ligation steps, but a complete system has been elusive. Here we have developed a NHEJ reconstitution system that includes the nuclease, polymerase, and ligase components to evaluate relative NHEJ efficiency and analyze ligated junctional sequences for various types of DNA ends, including blunt, 5′ overhangs, and 3′ overhangs. We find that different dsDNA end structures have differential dependence on these enzymatic components. The dependence of some end joining on only Ku and XRCC4·DNA ligase IV allows us to formulate a physical model that incorporates nuclease and polymerase components as needed.     

Non-homologous DNA end joining

DNA double strand breaks (DSB) are the most serious form of DNA damage. Repair of DSBs is important to prevent chromosomal fragmentation, translocations and deletions. Non-homologous end joining (NHEJ) is one of three major pathways for the repair of DSBs in human cells. In this process two DNA ends are joined directly, usually with no sequence homology, although in the case of same polarity of the single stranded overhangs in DSBs, regions of microhomology are utilized. NHEJ is typically imprecise, a characteristic that is useful for immune diversification in lymphocytes in V(D)J recombination. The main components of the NHEJ system in eukaryotes are the catalytic subunit of DNA protein kinase (DNA-PKcs), Ku proteins, XRCC4, DNA ligase IV, and Artemis. This review focuses on the mechanisms an dregulation of DSB repair by NHEJ in mammalian cells.