Role of polynucleotide kinase/phosphatase in mitochondrial DNA repair

Mutations in mitochondrial DNA (mtDNA) are implicated in a broad range of human diseases and in aging. Compared to nuclear DNA, mtDNA is more highly exposed to oxidative damage due to its proximity to the respiratory chain and the lack of protection afforded by chromatin-associated proteins. While repair of oxidative damage to the bases in mtDNA through the base excision repair pathway has been well studied, the repair of oxidatively induced strand breaks in mtDNA has been less thoroughly examined. Polynucleotide kinase/phosphatase (PNKP) processes strand-break termini to render them chemically compatible for the subsequent action of DNA polymerases and ligases. Here, we demonstrate that functionally active full-length PNKP is present in mitochondria as well as nuclei. Downregulation of PNKP results in an accumulation of strand breaks in mtDNA of hydrogen peroxide-treated cells. Full restoration of repair of the H(2)O(2)-induced strand breaks in mitochondria requires both the kinase and phosphatase activities of PNKP. We also demonstrate that PNKP contains a mitochondrial-targeting signal close to the C-terminus of the protein. We further show that PNKP associates with the mitochondrial protein mitofilin. Interaction with mitofilin may serve to translocate PNKP into mitochondria.     

Association of XRCC1 and tyrosyl DNA phosphodiesterase (Tdp1) for the repair of topoisomerase I-mediated DNA lesions

DNA topoisomerase I (Top1) is converted into a cellular poison by camptothecin (CPT) and various endogenous and exogenous DNA lesions. In this study, we used X-ray repair complementation group 1 (XRCC1)-deficient and XRCC1-complemented EM9 cells to investigate the mechanism by which XRCC1 affects the cellular responses to Top1 cleavage complexes induced by CPT. XRCC1 complementation enhanced survival to CPT-induced DNA lesions produced independently of DNA replication. CPT-induced comparable levels of Top1 cleavage complexes (single-strand break (SSB) and DNA-protein cross-links (DPC)) in both XRCC1-deficient and XRCC1-complemented cells. However, XRCC1-complemented cells repaired Top1-induced DNA breaks faster than XRCC1-deficient cells, and exhibited enhanced tyrosyl DNA phosphodiesterase (Tdp1) and polynucleotide kinase phosphatase (PNKP) activities. XRCC1 immunoprecipitates contained Tdp1 polypeptide, and both Tdp1 and PNKP activities, indicating a functional connection between the XRCC1 single-strand break repair pathway and the repair of Top1 covalent complexes by Tdp1 and PNKP.     

The Interaction between Polynucleotide Kinase Phosphatase and the DNA Repair Protein XRCC1 Is Critical for Repair of DNA Alkylation Damage and Stable Association at DNA Damage Sites

XRCC1 plays a key role in the repair of DNA base damage and single-strand breaks. Although it has no known enzymatic activity, XRCC1 interacts with multiple DNA repair proteins and is a subunit of distinct DNA repair protein complexes. Here we used the yeast two-hybrid genetic assay to identify mutant versions of XRCC1 that are selectively defective in interacting with a single protein partner. One XRCC1 mutant, A482T, that was defective in binding to polynucleotide kinase phosphatase (PNKP) not only retained the ability to interact with partner proteins that bind to different regions of XRCC1 but also with aprataxin and aprataxin-like factor whose binding sites overlap with that of PNKP. Disruption of the interaction between PNKP and XRCC1 did not impact their initial recruitment to localized DNA damage sites but dramatically reduced their retention there. Furthermore, the interaction between PNKP and the DNA ligase IIIα-XRCC1 complex significantly increased the efficiency of reconstituted repair reactions and was required for complementation of the DNA damage sensitivity to DNA alkylation agents of xrcc1 mutant cells. Together our results reveal novel roles for the interaction between PNKP and XRCC1 in the retention of XRCC1 at DNA damage sites and in DNA alkylation damage repair.     

PNKP在DNA损伤修复中的作用

多聚核苷酸激酶/磷酸酶(polynucleotide kinase/phosphatase,PNKP)是一种DNA末端修复酶,同时具有激酶和磷酸酶活性,在DNA单链断裂修复途径、碱基切除修复途径以及DNA双链断裂修复中的非同源末端连接途径中发挥着至关重要的作用。近年来,由于一种与PNKP相关的常染色体隐性遗传病——MCSZ综合征的发现,使得人们对PNKP的关注度进一步增加。笔者从与PNKP相互作用的X射线交叉互补修复基因1(X-ray repair cross-complementing group 1,XRCC1)、X射线交叉互补修复基因4(X-ray repair cross-complementing group 4,XRCC4)和毛细血管扩张性共济失调突变基因(ataxia-telangiectasia mutated,ATM)入手,对PNKP在DNA损伤修复中的作用进行概述。     

Dual Modes of Interaction between XRCC4 and Polynucleotide Kinase/Phosphatase: IMPLICATIONS FOR NONHOMOLOGOUS END JOINING*

XRCC4 plays a crucial role in the nonhomologous end joining (NHEJ) pathway of DNA double-strand break repair acting as a scaffold protein that recruits other NHEJ proteins to double-strand breaks. Phosphorylation of XRCC4 by protein kinase CK2 promotes a high affinity interaction with the forkhead-associated domain of the end-processing enzyme polynucleotide kinase/phosphatase (PNKP). Here we reveal that unphosphorylated XRCC4 also interacts with PNKP through a lower affinity interaction site within the catalytic domain and that this interaction stimulates the turnover of PNKP. Unexpectedly, CK2-phosphorylated XRCC4 inhibited PNKP activity. Moreover, the XRCC4·DNA ligase IV complex also stimulated PNKP enzyme turnover, and this effect was independent of the phosphorylation of XRCC4 at threonine 233. Our results reveal that CK2-mediated phosphorylation of XRCC4 can have different effects on PNKP activity, with implications for the roles of XRCC4 and PNKP in NHEJ.     

Phosphorylation of polynucleotide kinase/ phosphatase by DNA-dependent protein kinase and ataxia-telangiectasia mutated regulates its association with sites of DNA damage

Human polynucleotide kinase/phosphatase (PNKP) is a dual specificity 5'-DNA kinase/3'-DNA phosphatase, with roles in base excision repair, DNA single-strand break repair and non-homologous end joining (NHEJ); yet precisely how PNKP functions in the repair of DNA double strand breaks (DSBs) remains unclear. We demonstrate that PNKP is phosphorylated by the DNA-dependent protein kinase (DNA-PK) and ataxia-telangiectasia mutated (ATM) in vitro. The major phosphorylation site for both kinases was serine 114, with serine 126 being a minor site. Ionizing radiation (IR)-induced phosphorylation of cellular PNKP on S114 was ATM dependent, whereas phosphorylation of PNKP on S126 required both ATM and DNA-PK. Inactivation of DNA-PK and/or ATM led to reduced PNKP at DNA damage sites in vivo. Cells expressing PNKP with alanine or aspartic acid at serines 114 and 126 were modestly radiosensitive and IR enhanced the association of PNKP with XRCC4 and DNA ligase IV; however, this interaction was not affected by mutation of PNKP phosphorylation sites. Purified PNKP protein with mutation of serines 114 and 126 had decreased DNA kinase and DNA phosphatase activities and reduced affinity for DNA in vitro. Together, our results reveal that IR-induced phosphorylation of PNKP by ATM and DNA-PK regulates PNKP function at DSBs.     

XRCC1-mediated repair of strand breaks independent of PNKP binding

Repair of DNA-protein crosslinks and oxidatively damaged DNA base lesions generates intermediates with nicks or gaps with abnormal and blocked 3′-phosphate and 5′-OH ends that prevent the activity of DNA polymerases and ligases. End cleaning in mammalian cells by Tdp1 and PNKP produces the conventional 3′-OH and 5′-phosphate DNA ends suitable for completion of repair. This repair function of PNKP is facilitated by its binding to the scaffold protein XRCC1, and phosphorylation of XRCC1 by CK2 at several consensus sites enables PNKP binding and recruitment to DNA damage. To evaluate this documented repair process, a phosphorylation mutant of XRCC1, designed to eliminate PNKP binding, was stably expressed in Xrcc1^−/− mouse fibroblast cells. Analysis of PNKP-GFP accumulation at micro-irradiation induced damage confirmed that the XRCC1 phosphorylation mutant failed to support efficient PNKP recruitment, whereas there was rapid recruitment in cells expressing wild-type XRCC1. Recruitment of additional fluorescently-tagged repair factors PARP-1-YFP, GFF-XRCC1, PNKP-GFP and Tdp1-GFP to micro-irradiation induced damage was assessed in wild-type XRCC1-expressing cells. PARP-1-YFP recruitment was best fit to two exponentials, whereas kinetics for the other proteins were fit to a single exponential. The similar half-times of recruitment suggest that XRCC1 may be recruited with other proteins possibly as a pre-formed complex. Xrcc1^−/− cells are hypersensitive to the DNA-protein cross-link inducing agent camptothecin (CPT) and the DNA oxidative agent H₂O₂ due in part to compromised PNKP-mediated repair. However, cells expressing the PNKP interaction mutant of XRCC1 demonstrated marked reversal of CPT hypersensitivity. This reversal represents XRCC1-dependent repair in the absence of the phosphorylation-dependent PNKP recruitment and suggests either an XRCC1-independent mechanism of PNKP recruitment or a functional back-up pathway for cleaning of blocked DNA ends.     

AP-site cleavage activity of tyrosyl-DNA phosphodiesterase 1

APE-independent base excision repair (BER) pathway plays an important role in the regulation of DNA repair mechanisms. In this study it has been found that recently discovered tyrosyl-DNA phosphodiesterase 1 (Tdp1) catalyzes the AP site cleavage reaction to generate breaks with the 3'- and 5'-phosphate termini. The removal of the 3'-phosphate is performed by polynucleotide kinase phosphatase (PNKP). Tdp1 is known to interact stably with BER proteins: DNA polymerase beta (Pol β), XRCC1, PARP1 and DNA ligase III. The data suggest a role of Tdp1 in the new APE-independent BER pathway in mammals.     

XRCC1 and DNA polymerase β in cellular protection against cytotoxic DNA single-strand breaks

Single-strand breaks （SSBs） can occur in cells either directly, or indirectly following initiation of base excision repair （BER）. SSBs generally have blocked termini lacking the conventional 5＇-phosphate and 3＇-hydroxyl groups and require further processing prior to DNA synthesis and ligation. XRCC1 is devoid of any known enzymatic activity, but it can physically interact with other proteins involved in all stages of the overlapping SSB repair and BER pathways, including those that conduct the rate-limiting end-tailoring, and in many cases can stimulate their enzymatic activities. XRCC1^-/- mouse fibroblasts are most hypersensitive to agents that produce DNA lesions repaired by monofunctional glycosylase-initiated BER and that result in formation of indirect SSBs. A requirement for the deoxyribose phosphate lyase activity of DNA polymerase β （pol β） is specific to this pathway, whereas pol β is implicated in gap-filling during repair of many types of SSBs. Elevated levels of strand breaks, and diminished repair, have been demonstrated in MMS- treated XRCC1^-/-, and to a lesser extent in pol β^-/- cell lines, compared with wild-type cells. Thus a strong correlation is observed between cellular sensitivity to MMS and the ability of cells to repair MMS-induced damage. Exposure of wild-type and polβ^-/- cells to an inhibitor of PARP activity dramatically potentiates MMS-induced cytotoxicity. XRCC1^-/- cells are also sensitized by PARP inhibition demonstrating that PARP-mediated poly（ADP-ribosyl）ation plays a role in modulation of cytotoxicity beyond recruitment of XRCC 1 to sites of DNA damage.     

XRCC1 phosphorylation by CK2 is required for its stability and efficient DNA repair

XRCC1 is a scaffold protein that interacts with several DNA repair proteins and plays a critical role in DNA base excision repair (BER). XRCC1 protein is in a tight complex with DNA ligase IIIα (Lig III) and this complex is involved in the ligation step of both BER and repair of DNA single strand breaks. The majority of XRCC1 has previously been demonstrated to exist in a phosphorylated form and cells containing mutant XRCC1, that is unable to be phosphorylated, display a reduced rate of single strand break repair. Here, in an unbiased assay, we demonstrate that the cytoplasmic form of the casein kinase 2 (CK2) protein is the major protein kinase activity involved in phosphorylation of XRCC1 in human cell extracts and that XRCC1 phosphorylation is required for XRCC1-Lig III complex stability. We demonstrate that XRCC1-Lig III complex containing mutant XRCC1, in which CK2 phosphorylation sites have been mutated, is unstable. We also find that a knockdown of CK2 by siRNA results in both reduced XRCC1 phosphorylation and stability, which also leads to a reduced amount of Lig III and accumulation of DNA strand breaks. We therefore propose that CK2 plays an important role in DNA repair by contributing to the stability of XRCC1-Lig III complex.     

ATM-mediated phosphorylation of polynucleotide kinase/phosphatase is required for effective DNA double-strand break repair

The cellular response to double-strand breaks (DSBs) in DNA is a complex signalling network, mobilized by the nuclear protein kinase ataxia-telangiectasia mutated (ATM), which phosphorylates many factors in the various branches of this network. A main question is how ATM regulates DSB repair. Here, we identify the DNA repair enzyme polynucleotide kinase/phosphatase (PNKP) as an ATM target. PNKP phosphorylates 5'-OH and dephosphorylates 3'-phosphate DNA ends that are formed at DSB termini caused by DNA-damaging agents, thereby regenerating legitimate ends for further processing. We establish that the ATM phosphorylation targets on human PNKP-Ser 114 and Ser 126-are crucial for cellular survival following DSB induction and for effective DSB repair, being essential for damage-induced enhancement of the activity of PNKP and its proper accumulation at the sites of DNA damage. These findings show a direct functional link between ATM and the DSB-repair machinery.     

XRCC1 stimulates polynucleotide kinase by enhancing its damage discrimination and displacement from DNA repair intermediates

Human polynucleotide kinase (hPNK) is required for processing and rejoining DNA strand break termini. The 5'-DNA kinase and 3'-phosphatase activities of hPNK can be stimulated by the "scaffold" protein XRCC1, but the mechanism remains to be fully elucidated. Using a variety of fluorescence techniques, we examined the interaction of hPNK with XRCC1 and substrates that model DNA single-strand breaks. hPNK binding to substrates with 5'-OH termini was only approximately 5-fold tighter than that to identical DNA molecules with 5'-phosphate termini, suggesting that hPNK remains bound to the product of its enzymatic activity. The presence of XRCC1 did not influence the binding of hPNK to substrates with 5'-OH termini, but sharply reduced the interaction of hPNK with DNA bearing a 5'-phosphate terminus. These data, together with kinetic data obtained at limiting enzyme concentration, indicate a dual function for the interaction of XRCC1 with hPNK. First, XRCC1 enhances the capacity of hPNK to discriminate between strand breaks with 5'-OH termini and those with 5'-phosphate termini; and second, XRCC1 stimulates hPNK activity by displacing hPNK from the phosphorylated DNA product.     

Human Xip1 (C2orf13) is a novel regulator of cellular responses to DNA strand breaks

DNA strand breaks arise continuously as the result of intracellular metabolism and in response to a multitude of genotoxic agents. To overcome such challenges to genomic stability, cells have evolved genome surveillance pathways that detect and repair damaged DNA in a coordinated fashion. Here we identify the previously uncharacterized human protein Xip1 (C2orf13) as a novel component of the checkpoint response to DNA strand breaks. Green fluorescent protein-tagged Xip1 was rapidly recruited to sites of DNA breaks, and this accumulation was dependent on a novel type of zinc finger motif located in the C terminus of Xip1. The initial recruitment kinetics of Xip1 closely paralleled that of XRCC1, a central organizer of single strand break (SSB) repair, and its accumulation was both delayed and sustained when the detection of SSBs was abrogated by inhibition of PARP-1. Xip1 and XRCC1 stably interacted through recognition of CK2 phosphorylation sites in XRCC1 by the Forkhead-associated (FHA) domain of Xip1, and XRCC1 was required to maintain steady-state levels of Xip1. Moreover, Xip1 was phosphorylated on Ser-116 by ataxia telangiectasia-mutated in response to ionizing radiation, further underscoring the potential importance of Xip1 in the DNA damage response. Finally, depletion of Xip1 significantly decreased the clonogenic survival of cells exposed to DNA SSB- or double strand break-inducing agents. Collectively, these findings implicate Xip1 as a new regulator of genome maintenance pathways, which may function to organize DNA strand break repair complexes at sites of DNA damage.     

The protein kinase CK2 facilitates repair of chromosomal DNA single-strand breaks

CK2 was the first protein kinase identified and is required for the proliferation and survival of mammalian cells. Here, we have identified an unanticipated role for CK2. We show that this essential protein kinase phosphorylates the scaffold protein XRCC1 and thereby enables the assembly and activity of DNA single-strand break repair protein complexes in vitro and at sites of chromosomal breakage. Moreover, we show that inhibiting XRCC1 phosphorylation by mutation of the CK2 phosphorylation sites or preventing CK2 activity using a highly specific inhibitor ablates the rapid repair of cellular DNA single-strand breaks by XRCC1. These data identify a direct role for CK2 in the repair of chromosomal DNA strand breaks and in maintaining genetic integrity.     

A cell cycle-specific requirement for the XRCC1 BRCT II domain during mammalian DNA strand break repair

XRCC1 protein is essential for viability in mammals and is required for efficient DNA single-strand break repair and genetic stability following DNA base damage. We report here that XRCC1-dependent strand break repair in G(1) phase of the cell cycle is abolished by mutations created within the XRCC1 BRCT domain that interact with DNA ligase III. In contrast, XRCC1-dependent DNA strand break repair in S phase is largely unaffected by these mutations. These data describe a cell cycle-specific role for a BRCT domain, and we conclude that the XRCC1-DNA ligase III complex is required for DNA strand break repair in G(1) phase of the cell cycle but is dispensable for this process in S phase. The S-phase DNA repair process can remove both strand breaks induced in S phase and those that persist from G(1) and can in part compensate for lack of repair in G(1). This process correlates with the appearance of XRCC1 nuclear foci that colocalize with Rad51 and may thus function in concert with homologous recombination.     

Enzyme action at 3' termini of ionizing radiation-induced DNA strand breaks

gamma-Irradiation of DNA in vitro produces two types of single strand breaks. Both types of strand breaks contain 5'-phosphate DNA termini. Some strand breaks contain 3'-phosphate termini, some contain 3'-phosphoglycolate termini (Henner, W.D., Rodriguez, L.O., Hecht, S. M., and Haseltine, W. A. (1983) J. Biol. Chem. 258, 711-713). We have studied the ability of prokaryotic enzymes of DNA metabolism to act at each of these types of gamma-ray-induced 3' termini in DNA. Neither strand breaks that terminate with 3'-phosphate nor 3'-phosphoglycolate are substrates for direct ligation by T4 DNA ligase. Neither type of gamma-ray-induced 3' terminus can be used as a primer for DNA synthesis by either Escherichia coli DNA polymerase or T4 DNA polymerase. The 3'-phosphatase activity of T4 polynucleotide kinase can convert gamma-ray-induced 3'-phosphate but not 3'-phosphoglycolate termini to 3'-hydroxyl termini that can then serve as primers for DNA polymerase. E. coli alkaline phosphatase is also unable to hydrolyze 3'-phosphoglycolate groups. The 3'-5' exonuclease actions of E. coli DNA polymerase I and T4 DNA polymerase do not degrade DNA strands that have either type of gamma-ray-induced 3' terminus. E. coli exonuclease III can hydrolyze DNA with gamma-ray-induced 3'-phosphate or 3'-phosphoglycolate termini or with DNase I-induced 3'-hydroxyl termini. The initial action of exonuclease III at 3' termini of ionizing radiation-induced DNA fragments is to remove the 3' terminal phosphate or phosphoglycolate to yield a fragment of the same nucleotide length that has a 3'-hydroxyl terminus. These results suggest that repair of ionizing radiation-induced strand breaks may proceed via the sequential action of exonuclease, DNA polymerase, and DNA ligase. The possible role of exonuclease III in repair of gamma-radiation-induced strand breaks is discussed.     

XRCC1 protects against the lethality of induced oxidative DNA damage in nondividing neural cells

XRCC1 is a critical scaffold protein that orchestrates efficient single-strand break repair (SSBR). Recent data has found an association of XRCC1 with proteins causally linked to human spinocerebellar ataxias—aprataxin and tyrosyl-DNA phosphodiesterase 1—implicating SSBR in protection against neuronal cell loss and neurodegenerative disease. We demonstrate herein that shRNA lentiviral-mediated XRCC1 knockdown in human SH-SY5Y neuroblastoma cells results in a largely selective increase in sensitivity of the nondividing (i.e. terminally differentiated) cell population to the redox-cycling agents, menadione and paraquat; this reduced survival was accompanied by an accumulation of DNA strand breaks. Using hypoxanthine–xanthine oxidase as the oxidizing method, XRCC1 deficiency affected both dividing and nondividing SH-SY5Y cells, with a greater effect on survival seen in the former case, suggesting that the spectrum of oxidative DNA damage created dictates the specific contribution of XRCC1 to cellular resistance. Primary XRCC1 heterozygous mouse cerebellar granule cells exhibit increased strand break accumulation and reduced survival due to increased apoptosis following menadione treatment. Moreover, knockdown of XRCC1 in primary human fetal brain neurons leads to enhanced sensitivity to menadione, as indicated by increased levels of DNA strand breaks relative to control cells. The cumulative results implicate XRCC1, and more broadly SSBR, in the protection of nondividing neuronal cells from the genotoxic consequences of oxidative stress.     

The region of XRCC1 which harbours the three most common nonsynonymous polymorphic variants, is essential for the scaffolding function of XRCC1

XRCC1 functions as a non-enzymatic, scaffold protein in single strand break repair (SSBR) and base excision repair (BER). Here, we examine different regions of XRCC1 for their contribution to the scaffolding functions of the protein. We found that the central BRCT1 domain is essential for recruitment of XRCC1 to sites of DNA damage and DNA replication. Also, we found that ectopic expression of the region from residue 166-436 partially rescued the methyl methanesulfonate (MMS) hypersensitivity of XRCC1-deficient EM9 cells, suggesting a key role for this region in mediating DNA repair. The three most common amino acid variants of XRCC1, Arg194Trp, Arg280His and Arg399Gln, are located within the region comprising the NLS and BRCT1 domains, and these variants may be associated with increased incidence of specific types of cancer. While we could not detect differences in the intra-nuclear localization or the ability to support recruitment of POLβ or PNKP to micro-irradiated sites for these variants relative to the conservative protein, we did observe lower foci intensity after micro-irradiation and a reduced stability of the foci with the Arg280His and Arg399Gln variants, respectively. Furthermore, when challenged with MMS or hydrogen peroxide, we detected small but consistent differences in the repair profiles of cells expressing these two variants in comparison to the conservative protein.     

Role of human DNA glycosylase Nei-like 2 (NEIL2) and single strand break repair protein polynucleotide kinase 3'-phosphatase in maintenance of mitochondrial genome

The repair of reactive oxygen species-induced base lesions and single strand breaks (SSBs) in the nuclear genome via the base excision (BER) and SSB repair (SSBR) pathways, respectively, is well characterize, and important for maintaining genomic integrity. However, the role of mitochondrial (mt) BER and SSBR proteins in mt genome maintenance is not completely clear. Here we show the presence of the oxidized base-specific DNA glycosylase Nei-like 2 (NEIL2) and the DNA end-processing enzyme polynucleotide kinase 3'-phosphatase (PNKP) in purified human mitochondrial extracts (MEs). Confocal microscopy revealed co-localization of PNKP and NEIL2 with the mitochondrion-specific protein cytochrome c oxidase subunit 2 (MT-CO2). Further, chromatin immunoprecipitation analysis showed association of NEIL2 and PNKP with the mitochondrial genes MT-CO2 and MT-CO3 (cytochrome c oxidase subunit 3); importantly, both enzymes also associated with the mitochondrion-specific DNA polymerase γ. In cell association of NEIL2 and PNKP with polymerase γ was further confirmed by proximity ligation assays. PNKP-depleted ME showed a significant decrease in both BER and SSBR activities, and PNKP was found to be the major 3'-phosphatase in human ME. Furthermore, individual depletion of NEIL2 and PNKP in human HEK293 cells caused increased levels of oxidized bases and SSBs in the mt genome, respectively. Taken together, these studies demonstrate the critical role of NEIL2 and PNKP in maintenance of the mammalian mitochondrial genome.     

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.