Nonhomologous DNA End Joining Research Articles

Cernunnos Interacts with the XRCC4·DNA-ligase IV Complex and Is Homologous to the Yeast Nonhomologous End-joining Factor Nej1

DNA double strand breaks are considered as the most harmful DNA lesions and are repaired by either homologous recombination or nonhomologous end joining (NHEJ). A new NHEJ factor, Cernunnos, has been identified, the defect of which leads to a severe immunodeficiency condition associated with microcephaly and other developmental defects in humans. This presentation is reminiscent to that of DNA-ligase IV deficiency and suggests a possible interplay between Cernunnos and the XRCC4 x DNA-ligase IV complex. We show here that Cernunnos physically interacts with the XRCC4 x DNA-ligase IV complex. Moreover, a combination of sensitive methods of sequence analysis revealed that Cernunnos can be associated with the XRCC4 family of proteins and that it corresponds to the genuine homolog of the yeast Nej1 protein. Altogether these results shed new lights on the last step, the DNA religation, of the NHEJ pathway.

A Versatile and Efficient Gene-Targeting System for Aspergillus nidulans

Aspergillus nidulans is an important experimental organism, and it is a model organism for the genus Aspergillus that includes serious pathogens as well as commercially important organisms. Gene targeting by homologous recombination during transformation is possible in A. nidulans, but the frequency of correct gene targeting is variable and often low. We have identified the A. nidulans homolog (nkuA) of the human KU70 gene that is essential for nonhomologous end joining of DNA in double-strand break repair. Deletion of nkuA (nkuA delta) greatly reduces the frequency of nonhomologous integration of transforming DNA fragments, leading to dramatically improved gene targeting. We have also developed heterologous markers that are selectable in A. nidulans but do not direct integration at any site in the A. nidulans genome. In combination, nkuA delta and the heterologous selectable markers make up a very efficient gene-targeting system. In experiments involving scores of genes, 90% or more of the transformants carried a single insertion of the transforming DNA at the correct site. The system works with linear and circular transforming molecules and it works for tagging genes with fluorescent moieties, replacing genes, and replacing promoters. This system is efficient enough to make genomewide gene-targeting projects feasible.

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.

Severe combined immunodeficiency and microcephaly in siblings with hypomorphic mutations in DNA ligase IV

DNA double-strand breaks (dsb) during V(D)J recombination of T and B lymphocyte receptor genes are resolved by the non-homologous DNA end joining pathway (NHEJ) including at least six factors: Ku70, Ku80, DNA-PK(cs), Artemis, Xrcc4, and DNA ligase IV (Lig4). Artemis and Lig4 are the only known V(D)J/NHEJ factors found deficient in human genetic disorders. Null mutations of the Artemis gene result in a complete absence of T and B lymphocytes and increased cellular sensitivity to ionizing radiations, causing radiosensitive-SCID. Mutations of Lig4 are exclusively hypomorphic and have only been described in six patients, four exhibiting mild immunodeficiency associated with microcephaly and developmental delay, while two patient had leukemia. Here we report a SCID associated with microcephaly caused by compound heterozygous hypomorphic mutations in Lig4. Residual activity of Lig4 in these patients is underscored by a normal pattern of TCR-alpha and -beta junctions in the T cells of the patients and a moderate impairment of V(D)J recombination as tested in vitro. These observations contrast with the severity of the clinical immunodeficiency, suggesting that Lig4 may have additional critical roles in lymphocyte survival beyond V(D)J recombination.

Hybrid joint formation in human V(D)J recombination requires nonhomologous DNA end joining

In V(D)J recombination, the RAG proteins bind at a pair of signal sequences adjacent to the V, D, or J coding regions and cleave the DNA, resulting in two signal ends and two hairpinned coding ends. The two coding ends are joined to form a coding joint, and the two signal ends are joined to form a signal joint; this joining is done by the nonhomologous DNA end joining (NHEJ) pathway. A recombinational alternative in which a signal end is recombined with a coding end can also occur in a small percentage of the V(D)J recombination events in murine and human cells, and these are called hybrids (or hybrid joints). Two mechanisms have been proposed for the formation of these hybrids. One mechanism is via NHEJ, after initial cutting by RAGs. The second mechanism does not rely on NHEJ, but rather invokes that the RAGs can catalyze joining of the signal to the hairpinned coding end, by using the 3′OH of the signal end as a nucleophile to attack the phosphodiester bonds of the hairpinned coding end. In the present study, we addressed the question of which type of hybrid joining occurs in a physiological environment, where standard V(D)J recombination presumably occurs and normal RAG proteins are endogenously expressed. We find that all hybrids in vivo require DNA ligase IV in human cells, which is the final component of the NHEJ pathway. Hence, hybrid joints rely on NHEJ rather than on the RAG complex for joining.

Inhibition of Non-Homologous End Joining and integration of DNA upon transformation of Rhizopus oryzae

Site-directed integration of DNA in the fungus Rhizopus has long been problematic because linearized plasmids used for transformation tend to replicate in high-molecular-weight concatenated structures, and rarely integrate into the chromosome. This work examines the methods that might interfere with the multimerization process, select against plasmids that had recircularized, and encourage strand invasion, hopefully leading to plasmid integration. In vitro methods were used to determine if the structure of the double-strand break had any effect on the ability to rejoin plasmid ends. In cell-free extracts, little difference in end-joining activity was found between linearized plasmids with 5' overhangs, 3' overhangs, or blunt ends. In addition, dephosphorylation of ends had no effect. Transformation of plasmids prepared in the same ways confirmed that they were easily religated in vivo, with almost all prototrophic isolates retaining autonomously replicated plasmids. It was possible to block religation by modifying the free ends of the linearized plasmids using oligonucleotide adapters which were blocked at the 3'-OH position and contained phosphorothioate nucleotides to make them nuclease-resistant. However, gene replacement, with repair of the auxotrophic mutation in the host chromosome, was the predominant event observed upon the transformation of these plasmids. The highest rates of integration were obtained with a plasmid containing a truncated, non-functional pyrG gene. Autonomous replication of this plasmid did not support prototrophic growth, but homologous recombination into the chromosome restored the function of the endogenous pyrG gene. All of the transformants obtained with this selective construct were found to have integrated the plasmid, with multicopy insertion being common.

Influence of double-strand-break repair pathways on radiosensitivity throughout the cell cycle in CHO cells

Unrepaired DNA double-strand breaks (DSBs) produced by ionizing radiation (IR) are a major determinant of cell killing. To determine the contribution of DNA repair pathways to the well-established cell cycle variation in IR sensitivity, we compared the radiosensitivity of wild-type CHO cells to mutant lines defective in nonhomologous end joining (NHEJ), homologous recombination repair (HRR), and the Fanconi anemia pathway. Cells were irradiated with IR doses that killed ∼90% of each asynchronous population, separated into synchronous fractions by centrifugal elutriation, and assayed for survival (colony formation). Wild-type cells had lowest resistance in early G1 and highest resistance in S phase, followed by declining resistance as cells move into G2/M. In contrast, HR-defective cells ( xrcc3 mutation) were most resistant in early G1 and became progressively less resistant in S and G2/M, indicating that the S-phase resistance in wild-type cells requires HRR. Cells defective in NHEJ ( dna- pk cs mutation) were exquisitely sensitive in early G1, most resistant in S phase, and then somewhat less resistant in G2/M. Fancg mutant cells had almost normal IR sensitivity and normal cell cycle dependence, suggesting that Fancg contributes modestly to survival and in a manner that is independent of cell cycle position.

The Artemis:DNA-PKcs endonuclease cleaves DNA loops, flaps, and gaps

In eukaryotic cells, nonhomologous DNA end joining (NHEJ) is a major pathway for repair of double-strand DNA breaks (DSBs). Artemis and the 469 kDa DNA-dependent protein kinase (DNA-PKcs) together form a key nuclease for NHEJ in vertebrate organisms. The structure-specific endonucleolytic activity of Artemis is activated by binding to and phosphorylation by DNA-PKcs. We tested various DNA structures in order to understand the range of structural features that are recognized by the Artemis:DNA-PKcs complex. We find that all tested substrates that contain single-to-double-strand transitions can be cleaved by the Artemis:DNA-PKcs complex near the transition region. The cleaved substrates include heterologous loops, stem-loops, flaps, and gapped substrates. Such versatile activity on single-/double-strand transition regions is important in understanding how reconstituted NHEJ systems that lack DNA polymerases can join incompatible DNA ends and yet preserve 3′ overhangs. Additionally, the flexibility of the Artemis:DNA-PKcs nuclease may be important in removing secondary structures that hinder processing of DNA ends during NHEJ.

Non-homologous DNA End Joining Repair in Normal and Leukemic Cells Depends on the Substrate Ends

Double-strand breaks (DSBs) are the most serious DNA damage which, if unrepaired or misrepaired, may lead to cell death, genomic instability or cancer transformation. In human cells they can be repaired mainly by non-homologous DNA end joining (NHEJ). The efficacy of NHEJ pathway was examined in normal human lymphocytes and K562 myeloid leukemic cells expressing the BCR/ABL oncogenic tyrosine kinase activity and lacking p53 tumor suppressor protein. In our studies we employed a simple and rapid in vitro DSB end joining assay based on fluorescent detection of repair products. Normal and cancer cells were able to repair DNA damage caused by restriction endonucleases, but the efficiency of the end joining was dependent on the type of cells and the structure of DNA ends. K562 cells displayed decreased NHEJ activity in comparison to normal cells for 5' complementary DNA overhang. For blunt-ended DNA there was no significant difference in end joining activity. Both kinds of cells were found about 10-fold more efficient for joining DNA substrates with compatible 5' overhangs than those with blunt ends. Our recent findings have shown that stimulation of DNA repair could be involved in the drug resistance of BCR/ABL-positive cells in anticancer therapy. For the first time the role of STI571 was investigated, a specific inhibitor of BCR/ABL oncogenic protein approved for leukemia treatment in the NHEJ pathway. Surprisingly, STI571 did not change the response of BCR/ABL-positive K562 cells in terms of NHEJ for both complementary and blunt ends. Our results suggest that the various responses of the cells to DNA damage via NHEJ can be correlated with the differences in the genetic constitution of human normal and cancer cells. However, the role of NHEJ in anticancer drug resistance in BCR/ABL-positive cells is questionable.

384. Double-Stranded DNA Breaks Stimulate the Frequency of Targeted Gene Repair in Mammalian Cells

We have reported that double-stranded breaks in DNA stimulate the gene repair activity of the homologous recombination pathway. This induction also leads to a stalling of DNA replication thereby slowing the cell cycle phase in which the gene repair reaction occurs most often. DNA cleavage was induced initially by using anticancer drugs like etoposide (VP16), camptothecin (CPT) or chemicals like hydroxyurea (HU) and methymethanesulfonate (MMS). The last of these agents, MMS, is an alkyaltion agent capable of inducing DNA lesions and subsequent single and double strand breaks (DSB). This damage activates the non-homologous end joining (NHEJ) and homologous recombination (HR) DNA repair pathways, a response that has been shown to increase the frequency of oligonucleotide-directed gene repair. We show in DLD-1 cells that the conversion frequency of a mutant base pair to wild-type in an integrated enhanced green fluorescent protein gene is increased by pretreatment with MMS. This stimulation is dose-dependent and correlates to the level of DSB induced by MMS treatment. These DSBs result in Rad51p nuclear-relocalization and foci formation, a specific marker for HR activation. Additionally the cells exhibit cell cycle delay in S phase due to the stalling of replication forks. Our data suggest that MMS-induced DNA damage elicits a cellular response that stimulates gene repair in mammalian cells.

The MRE11-RAD50-XRS2 Complex, in Addition to Other Non-homologous End-joining Factors, Is Required for V(D)J Joining in Yeast

Lymphoid cells of the vertebrate immune system rely on factors in the non-homologous end-joining (NHEJ) DNA repair pathway to form signal joints during V(D)J recombination. Unlike other end-joining reactions, signal joint formation is a specialized case of NHEJ that also requires the lymphoid-specific RAG proteins. Whether V(D)J recombination requires the Mre11-Rad50-Nbs1 complex remains an open question, as null mutations in any member of the complex are lethal in mammals. However, Saccharomyces cerevisiae strains carrying null mutations in components of the homologous Mre11p-Rad50p-Xrs2p (MRX) complex are viable. We therefore took advantage of a recently developed V(D)J recombination assay in yeast to assess the role of MRX in V(D)J joining. Here we confirmed that signal joint formation in yeast is dependent on the same NHEJ factors known to be required in mammalian cells. In addition, we showed an absolute requirement for the MRX complex in signal joining, suggesting that the Mre11-Rad50-Nbs1 complex may be required for signal joint formation in mammalian cells as well.

Evaluation of Thermal Cisplatin Sensitization in Chicken DT40 Cells, Normal and Deficient, in DNA Homologous and Nonhomologous Endjoining Repair

Evaluation of Thermal Cisplatin Sensitization in Chicken DT40 Cells, Normal and Deficient, in DNA Homologous and Nonhomologous Endjoining Repair

Inhibition of homologous recombination by variants of the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs)

Two major DNA double-strand break repair pathways exist in all eukaryotes, nonhomologous DNA end joining (NHEJ) and homologous recombination (HR). Although both pathways can function throughout the cell cycle, NHEJ predominates in G0/G1) (when a replicated sister chromatid is unavailable), whereas HR makes a more substantial contribution in S and G2. How a cell chooses between these two important DNA repair pathways is largely unknown. DNA-dependent protein kinase (DNA-PK) is critical for NHEJ. Here, we describe two conserved splice variants of a catalytic subunit of DNA-PK (DNA-PKcs) that are expressed predominately in nondividing cells. Although both encode stable products, neither reverses the NHEJ defects in DNA-PKcs-deficient cells. In fact, cells expressing one of the DNA-PKcs variants are slightly more radiosensitive than cells completely deficient in DNA-PKcs. We investigated whether cells expressing the DNA-PKcs variants had any other DNA repair deficits and found that these cells are considerably more sensitive to both etoposide and mitomycin C than cells that express no DNA-PKcs at all. Because repair of DNA damage induced by these two agents requires intact HR, we tested whether the NHEJ-defective variants of DNA-PKcs inhibit double-strand break-induced HR in an integrated substrate. In cells expressing the NHEJ-defective variants, HR was markedly reduced. Because the splice variants are expressed highly only in nondividing cells, quiescent cells would be afforded a mechanism to inhibit repair by means of HR when sister chromatids are not available as templates for accurate repair with low risk of genome rearrangement, thereby enhancing genome stability.

DNA damage and nonhomologous end joining in excitotoxicity: Neuroprotective role of DNA-PKcs in kainic acid-induced seizures

DNA repair plays a critical, but imprecisely defined role in excitotoxic injury and neuronal survival throughout adulthood. We utilized an excitotoxic injury model to compare the location and phenotype of degenerating neurons in mice (strain 129-C57BL) deficient in the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), an enzyme required for nonhomologous end joining (NHEJ). Brains from untreated adult heterozygous and DNA-PKcs null mice displayed comparable cytoarchitecture and undetectable levels of cell death. By day 1, and extending through 4 days following kainic acid-induced seizures, brains from DNA-PKcs null mice showed widespread neurodegeneration that encompassed the entire hippocampal CA1-CA3 pyramidal cell layer, entorhinal cortex, and lateral septum, with relative sparing of the dentate gyrus granule cell layer and hilus, as judged by toluidine blue, Fluoro-Jade B, and terminal dUTP nick end labeling staining. In contrast, seizure-related neurodegeneration in heterozygous littermates was limited to the CA3 region of the hippocampus. NeuN and calbindin staining revealed a selective decrease in the number and density of NeuN-positive neurons in the pyramidal layers of degenerating regions in both heterozygous and DNA-PKcs null mice. To elucidate the mechanisms leading to cell death, we examined an involvement of the p53 pathway, known to be induced by DNA damage. Addition of pifithrin-alpha, a p53 inhibitor, or expression of a dominant-negative p53 rescued neurons from kainate-induced excitotoxic cell death in primary cortical cultures derived from wildtype, DNA-PKcs heterozygous, or DNA-PKcs null neonatal mice. Moreover, pifithrin-alpha prevented kainate-induced loss of mitochondrial membrane potential, dendrite degeneration, and cell death. Results suggest that NHEJ plays a neuroprotective role in excitotoxicity, within the perforant, Schaffer collateral, hippocampal-septal, and temperoammonic pathways, in part by repairing DNA damage that would otherwise result in activation of a p53-dependent apoptotic cascade.

The Pathway for the Production of Inositol Hexakisphosphate in Human Cells

The yeast and Drosophila pathways leading to the production of inositol hexakisphosphate (InsP(6)) have been elucidated recently. The in vivo pathway in humans has been assumed to be similar. Here we show that overexpression of Ins(1,3,4)P(3) 5/6-kinase in human cell lines results in an increase of inositol tetrakisphosphate (InsP(4)) isomers, inositol pentakisphosphate (InsP(5)) and InsP(6), whereas its depletion by RNA interference decreases the amounts of these inositol phosphates. Expression of Ins(1,3,4,6)P(4) 5-kinase does not increase the amount of InsP(5) and InsP(6), although its depletion does block InsP(5) and InsP(6) production, showing that it is necessary for production of InsP(5) and InsP(6). Expression of Ins(1,3,4,5,6)P(5) 2-kinase increases the amount of InsP(6) by depleting the InsP(5) in the cell, and depletion of 2-kinase decreases the amount of InsP(6) and causes an increase in InsP(5). These results are consistent with a pathway that produces InsP(6) through the sequential action of Ins(1,3,4)P(3) 5/6-kinase, Ins(1,3,4,6)P(4) 5-kinase, and Ins(1,3,4,5,6)P5 2-kinase to convert Ins(1,3,4)P(3) to InsP(6). Furthermore, the evidence implicates 5/6-kinase as the rate-limiting enzyme in this pathway.

A Role for Rat Inositol Polyphosphate Kinases rIPK2 and rIPK1 in Inositol Pentakisphosphate and Inositol Hexakisphosphate Production in Rat-1 Cells

Over 30 inositol polyphosphates are known to exist in mammalian cells; however, the majority of them have uncharacterized functions. In this study we investigated the molecular basis of synthesis of highly phosphorylated inositol polyphosphates (such as inositol tetrakisphosphate, inositol pentakisphosphate (IP5), and inositol hexakisphosphate (IP6)) in rat cells. We report that heterologous expression of rat inositol polyphosphate kinases rIPK2, a dual specificity inositol trisphosphate/inositol tetrakisphosphate kinase, and rIPK1, an IP5 2-kinase, were sufficient to recapitulate IP6 synthesis from inositol 1,4,5-trisphosphate in mutant yeast cells. Overexpression of rIPK2 in Rat-1 cells increased inositol 1,3,4,5,6-pentakisphosphate (I(1,3,4,5,6)P5) levels about 2-3-fold compared with control. Likewise in Rat-1 cells, overexpression of rIPK1 was capable of completely converting I(1,3,4,5,6)P5 to IP6. Simultaneous overexpression of both rIPK2 and rIPK1 in Rat-1 cells increased both IP5 and IP6 levels. To reduce IPK2 activity in Rat-1 cells, we introduced vector-based short interference RNA against rIPK2. Cells harboring the short interference RNA had a 90% reduction of mRNA levels and a 75% decrease of I(1,3,4,5,6)P5. These data confirm the involvement of IPK2 and IPK1 in the conversion of inositol 1,4,5-trisphosphate to IP6 in rat cells. Furthermore these data suggest that rIPK2 and rIPK1 act as key determining steps in production of IP5 and IP6, respectively. The ability to modulate the intracellular inositol polyphosphate levels by altering IPK2 and IPK1 expression in rat cells will provide powerful tools to study the roles of I(1,3,4,5,6)P5 and IP6 in cell signaling.

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.

Polyethyleneimine grafted with pluronic P85 enhances Ku86 antisense delivery and the ionizing radiation treatment efficacy in vivo

In an effort to improve the efficacy of antisense delivery, we evaluated polyethyleneimine (PEI, 2 kDa) alone or grafted with nonionic amphiphilic block copolymer Pluronic (P85) as a carrier for Ku86 antisense oligonucleotide (ASO) delivery. Ku86 is an abundant nuclear protein that plays an important role in nonhomologous DNA end joining and has implications in tumorigenesis and acquired drug resistance. Transfection of adherent and suspension cell lines with Ku86 ASOs complexed with P85-g-PEI (2 kDa) conjugates was associated with a specific decrease in Ku86 mRNA levels (EC50<75 nM and EC50<250 nM, respectively, n=3). More importantly, no requirement for reduced serum conditions was necessary during transfection. In contrast, whereas Ku86 ASOs complexed with PEI (2 kDa) alone were effective in decreasing Ku86 mRNA levels in adherent cell lines (EC50<75 nM, n=3), the formulation did not produce any detectable decrease in Ku86 mRNA levels in suspension cell lines. Transfection of adherent cell lines with 500 nM Ku86 ASOs formulated with P85-g-PEI (2 kDa) was associated with a specific decrease (<10% remaining of control) in Ku86 protein expression and a two-fold increased cell death after treatment with ionizing radiation (IR). In athymic nude mice bearing subcutaneous human HT29 colon adenocarcinoma xenografts, Ku86 ASO-P85-g-PEI (2 kDa) administration (15 mg/kg, subcutaneously) with a Q1D x 7 treatment schedule, when combined with a single dose of IR (6 Gy), caused a significant inhibition of HT29 tumor growth compared with mismatch- and naked antisense-pretreated control groups (time from 200 to 1000 mm3, 126.9 versus 84.18 and 87.76 days, P<0.005). A potentiation of the antitumor activity was observed in all mice treated with Ku86 ASO-P85-g-PEI (2 kDa) formulation; however, tumor growth inhibition was reversible upon treatment cessation. No morbidity/mortality or changes in histopathology were observed under this treatment regiment. Our results indicate that P85-g-PEI (2 kDa) conjugates may increase the efficacy of Ku86 ASO delivery in management of resistant malignancies, thus providing a rationale for their evaluation in cancer patients in combination with conventional anticancer therapies.

Fluorescence Anisotropy Studies on the Ku-DNA Interaction: ANION AND CATION EFFECTS

DNA non-homologous end joining starts with the binding of Ku heterodimers to double strand breaks. In this work, we characterized the thermodynamics of the Ku-DNA interaction by fluorescence anisotropy of the probe-labeled DNA. We determined that the microscopic dissociation constant (kd) for the binding of Ku to a DNA binding site of the proper length (>20 bp) ranges from 22 to 29 nm at 300 mm NaCl. The binding isotherms for DNA duplexes with two or three heterodimers were analyzed with two independent models considering the presence and absence of overlapping binding sites. This analysis demonstrated that there is no or very weak nearest-neighbor cooperativity among the Ku molecules. These models can most likely be applied to study the interaction of Ku with duplexes of any length. Furthermore, our salt dependence studies indicated that electrostatic interactions play a major role in the binding of Ku to DNA and that the kd decreases approximately 60-fold as the salt concentration is lowered from 300 to 200 mm. The slope (Gammasalt) of the plot of log kd versus log[NaCl] is 12.4 +/- 0.1. This value is among the highest reported in the literature for a protein-DNA interaction and suggests that approximately 12 ions are released upon formation of the Ku-DNA complex. In addition, comparison of the slope values measured upon varying the type of cation and anion indicated that approximately nine cations and three anions are released from DNA and Ku, respectively, when the complex is formed.

Xrcc4 physically links DNA end processing by polynucleotide kinase to DNA ligation by DNA ligase IV.

Nonhomologous end joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammalian cells. A critical step in this process is DNA ligation, involving the Xrcc4-DNA ligase IV complex. DNA end processing is often a prerequisite for ligation, but the coordination of these events is poorly understood. We show that polynucleotide kinase (PNK), with its ability to process ionizing radiation-induced 5'-OH and 3'-phosphate DNA termini, functions in NHEJ via an FHA-dependent interaction with CK2-phosphorylated Xrcc4. Analysis of the PNK FHA-Xrcc4 interaction revealed that the PNK FHA domain binds phosphopeptides with a unique selectivity among FHA domains. Disruption of the Xrcc4-PNK interaction in vivo is associated with increased radiosensitivity and slower repair kinetics of DSBs, in conjunction with a diminished efficiency of DNA end joining in vitro. Therefore, these results suggest a new role for Xrcc4 in the coordination of DNA end processing with DNA ligation.