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.     

A mechanism for DNA-PK activation requiring unique contributions from each strand of a DNA terminus and implications for microhomology-mediated nonhomologous DNA end joining

DNA-dependent protein kinase (DNA-PK) is an essential component of the nonhomologous end joining pathway (NHEJ), responsible for the repair of DNA double-strand breaks. Ku binds a DSB and recruits the catalytic subunit, DNA-PKcs, where it is activated once the kinase is bound to the DSB. The precise mechanism by which DNA activates DNA-PK remains unknown. We have investigated the effect of DNA structure on DNA-PK activation and results demonstrate that in Ku-dependent DNA-PKcs reactions, DNA-PK activation with DNA effectors containing two unannealed ends was identical to activation observed with fully duplex DNA effectors of the same length. The presence of a 6-base single-stranded extension resulted in decreased activation compared to the fully duplex DNA. DNA-PK activation using DNA effectors with compatible termini displayed increased activity compared to effectors with noncompatible termini. A strand orientation preference was observed in these reactions and suggests a model where the 3' strand of the terminus is responsible for annealing and the 5' strand is involved in activation of DNA-PK. These results demonstrate the influence of DNA structure and orientation on DNA-PK activation and provide a molecular mechanism of activation resulting from compatible termini, an essential step in microhomology-mediated NHEJ.     

Non-homologous end-joining defect in fanconi anemia hematopoietic cells exposed to ionizing radiation

Fanconi anemia is a genetically heterogeneous recessive disease characterized mainly by bone marrow failure and cancer predisposition. Although it is accepted that Fanconi cells are highly sensitive to DNA crosslinking agents, their response to ionizing radiation is still unclear. Using pulsed-field gel electrophoresis, we have observed that radiation generates a similar number of DNA double-strand breaks in normal and Fanconi cells from three (FA-A, FA-C and FA-F) of the 11 complementation groups identified. Nonsynchronized as well as nonproliferating Fanconi anemia cells showed an evident defect in rejoining the double-strand breaks generated by ionizing radiation, indicating defective non-homologous end-joining repair. At the cellular level, no difference in the radiosensitivity of normal and FA-A lymphoblast cells was noted, and a modest increase in the radiosensitivity of Fanca-/- hematopoietic progenitor cells was observed compared to Fanca+/+ cells. Finally, when animals were exposed to a fractionated total-body irradiation of 5 Gy, a similar hematopoietic syndrome was observed in wild-type and Fanca-/- mice. Taken together, our observations suggest that Fanconi cells, in particular those having nonfunctional Fanconi proteins upstream of FANCD2, have a defect in the non-homologous end-joining repair of double-strand breaks produced by ionizing radiation, and that compensatory mechanisms of DNA repair and/or stem cell regeneration should limit the impact of this defect in irradiated organisms.     

Interplay between Ku, Artemis, and the DNA-dependent protein kinase catalytic subunit at DNA ends

Repair of DNA double strand breaks (DSB) by the nonhomologous end-joining pathway in mammals requires at least seven proteins involved in a simplified two-step process: (i) recognition and synapsis of the DNA ends dependent on the DNA-dependent protein kinase (DNA-PK) formed by the Ku70/Ku80 heterodimer and the catalytic subunit DNA-PKcs in association with Artemis; (ii) ligation dependent on the DNA ligase IV.XRCC4.Cernunnos-XLF complex. The Artemis protein exhibits exonuclease and endonuclease activities that are believed to be involved in the processing of a subclass of DSB. Here, we have analyzed the interactions of Artemis and nonhomologous end-joining pathway proteins both in a context of human nuclear cell extracts and in cells. DSB-inducing agents specifically elicit the mobilization of Artemis to damaged chromatin together with DNA-PK and XRCC4/ligase IV proteins. DNA-PKcs is necessary for the loading of Artemis on damaged DNA and is the main kinase that phosphorylates Artemis in cells damaged with highly efficient DSB producers. Under kinase-preventive conditions, both in vitro and in cells, Ku-mediated assembly of DNA-PK on DNA ends is responsible for a dissociation of the DNA-PKcs.Artemis complex. Conversely, DNA-PKcs kinase activity prevents Artemis dissociation from the DNA-PK.DNA complex. Altogether, our data allow us to propose a model in which a DNA-PKcs-mediated phosphorylation is necessary both to activate Artemis endonuclease activity and to maintain its association with the DNA end site. This tight functional coupling between the activation of both DNA-PKcs and Artemis may avoid improper processing of DNA.     

Protein phosphatases regulate DNA-dependent protein kinase activity

DNA-dependent protein kinase (DNA-PK) is a complex of DNA-PK catalytic subunit (DNA-PKcs) and the DNA end-binding Ku70/Ku80 heterodimer. DNA-PK is required for DNA double strand break repair by the process of nonhomologous end joining. Nonhomologous end joining is a major mechanism for the repair of DNA double strand breaks in mammalian cells. As such, DNA-PK plays essential roles in the cellular response to ionizing radiation and in V(D)J recombination. In vitro, DNA-PK undergoes phosphorylation of all three protein subunits (DNA-PK catalytic subunit, Ku70 and Ku80) and phosphorylation correlates with inactivation of the serine/threonine protein kinase activity of DNA-PK. Here we show that phosphorylation-induced loss of the protein kinase activity of DNA-PK is restored by the addition of the purified catalytic subunit of either protein phosphatase 1 or protein phosphatase 2A (PP2A) and that this reactivation is blocked by the potent protein phosphatase inhibitor, microcystin. We also show that treating human lymphoblastoid cells with either okadaic acid or fostriecin, at PP2A-selective concentrations, causes a 50-60% decrease in DNA-PK protein kinase activity, although the protein phosphatase 1 activity in these cells was unaffected. In vivo phosphorylation of DNA-PKcs, Ku70, and Ku80 was observed when cells were labeled with [(32)P]inorganic phosphate in the presence of the protein phosphatase inhibitor, okadaic acid. Together, our data suggest that reversible protein phosphorylation is an important mechanism for the regulation of DNA-PK protein kinase activity and that the protein phosphatase responsible for reactivation in vivo is a PP2A-like enzyme.     

A Ku80 fragment with dominant negative activity imparts a radiosensitive phenotype to CHO-K1 cells

DNA non-homologous end joining, the major mechanism for the repair of DNA double-strands breaks (DSB) in mammalian cells requires the DNA-dependent protein kinase (DNA-PK), a complex composed of a large catalytic subunit of 460 kDa (DNA-PKcs) and the heterodimer Ku70–Ku80 that binds to double-stranded DNA ends. Mutations in any of the three subunits of DNA-PK lead to extreme radiosensitivity and DSB repair deficiency. Here we show that the 283 C-terminal amino acids of Ku80 introduced into the Chinese hamster ovary cell line CHO-K1 have a dominant negative effect. Expression of Ku(449–732) in CHO cells was verified by northern blot analysis and resulted in decreased Ku-dependent DNA end-binding activity, a diminished capacity to repair DSBs as determined by pulsed field gel electrophoresis and decreased radioresistance determined by clonogenic survival. The stable modifications observed at the molecular and cellular level suggest that this fragment of Ku80 confers a dominant negative effect providing an important mechanism to sensitise radioresistant cells.     

Expression of DNA-dependent protein kinase in human granulocytes

Human polymorphonuclear leukocytes (PMN) have been reported to completely lack of DNA-dependent protein kinase (DNA-PK) which is composed of Ku protein and the catalytic subunit DNA-PKcs, needed for nonhomologous end-joining (NHEJ) of DNA double-strand breaks. Promyelocytic HL-60 cells express a variant form of Ku resulting in enhanced radiation sensitivity. This raises the question if low efficiency of NHEJ, instrumental for the cellular repair of oxidative damage, is a normal characteristic of myeloid differentiation. Here we confirmed the complete lack of DNAPK in PMN protein extracts, and the expression of the truncated Ku86 variant form in HL-60. However, this degradation of DNA-PK was shown to be due to a DNA-PK-degrading protease in PMN and HL-60. In addition, by using a protease-resistant whole cell assay, both Ku86 and DNA-PKcs could be demonstrated in PMN, suggesting the previously reported absence in PMN of DNA-PK to be an artefact. The levels of Ku86 and DNA-PKcs were much reduced in PMN, as compared with that of the lymphocytes, whereas HL-60 displayed a markedly elevated DNA-PK concentration. In conclusion, our findings provide evidence of reduced, not depleted expression of DNA-PK during the mature stages of myeloid differentiation.     

Interaction of the Ku heterodimer with the DNA ligase IV/Xrcc4 complex and its regulation by DNA-PK

DNA non-homologous end-joining (NHEJ) is a major mechanism for repairing DNA double-stranded (ds) breaks in mammalian cells. Here, we characterize the interaction between two key components of the NHEJ machinery, the Ku heterodimer and the DNA ligase IV/Xrcc4 complex. Our results demonstrate that Ku interacts with DNA ligase IV via its tandem BRCT domain and that this interaction is enhanced in the presence of Xrcc4 and dsDNA. Moreover, residues 644-748 of DNA ligase IV encompassing the first BRCT motif are necessary for binding. We show that Ku needs to be in its heterodimeric form to bind DNA ligase IV and that the C-terminal tail of Ku80, which mediates binding to DNA-PKcs, is dispensable for DNA ligase IV recognition. Although the interaction between Ku and DNA ligase IV/Xrcc4 occurs in the absence of DNA-PKcs, the presence of the catalytic subunit of DNA-PK kinase enhances complex formation. Previous studies have shown that DNA-PK kinase activity causes disassembly of DNA-PKcs from Ku at the DNA end. Here, we show that DNA-PK kinase activity also results in disassembly of the Ku/DNA ligase IV/Xrcc4 complex. Collectively, our findings provide novel information on the protein-protein interactions that regulate NHEJ in cells.     

Analysis of DNA-dependent protein kinase-mediated DNA end joining by two-photon fluorescence cross-correlation spectroscopy

Nonhomologous end joining (NHEJ) is the primary mechanism by which mammalian cells repair DNA double-strand breaks (DSBs). Proteins known to play a role in NHEJ include the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), the Ku 70/Ku 80 heterodimer (Ku), XRCC4, and DNA ligase IV. One of the main roles of the DNA-PKcs-Ku complex is to bring the ends of the DSB together in a process termed synapsis, prior to end joining. Synapsis results in the autophosphorylation of DNA-PKcs, which is required to make the DNA ends available for ligation. Here, we describe a novel assay using two-photon fluorescence cross-correlation spectroscopy that allows for the analysis of DNA synapsis and end joining in solution using purified proteins. We demonstrate that although autophosphorylation-defective DNA-PKcs does not support DNA ligase-mediated DNA end joining, like wild-type (WT) DNA-PKcs, it is capable of Ku-dependent DNA synapsis in solution. Moreover, we show that, in the presence of Ku, both WT DNA-PKcs and autophosphorylation-defective DNA-PKcs promote the formation of multiple, large multi-DNA complexes in solution, suggesting that, rather than align two opposing DNA ends, multiple DNA-PK molecules may serve to bring multiple DNA ends into the NHEJ complex.     

Effect of Ku86 and DNA-PKcs deficiency on non-homologous end-joining and homologous recombination using a transient transfection assay

In mammalian cells, DNA double-strand breaks are repaired by non-homologous end-joining and homologous recombination, both pathways being essential for the maintenance of genome integrity. We determined the effect of mutations in Ku86 and DNA-PK on the efficiency and the accuracy of double-strand break repair by non-homologous end-joining and homologous recombination in mammalian cells. We used an assay, based on the transient transfection of a linearized plasmid DNA, designed to simultaneously detect transfection and recombination markers. In agreement with previous results non-homologous end-joining was largely compromised in Ku86 deficient cells, and returned to normal in the Ku86-complemented isogenic cell line. In addition, analysis of DNA plasmids recovered from Ku86 mutant cells showed an increased use of microhomologies at the nonhomologous end joining junctions, and displayed a significantly higher frequency of DNA insertions compared to control cells. On the other hand, the DNA-PKcs deficient cell lines showed efficient double-strand break repair by both mechanisms.     

Role of DNA-PK subunits in radiosensitization by hyperthermia.

Thermal radiosensitization is thought to result from inhibition of repair of radiation-induced DNA damage, DNA double-strand breaks in particular. Since the DNA-dependent protein kinase (DNA-PK) complex plays a major role in the nonhomologous end-joining of DSBs, it has been suggested that inactivation of this complex as a whole or of its individual subunits by heat might be involved in radiosensitization by heat. To test this hypothesis further, the ability of heat to enhance the radiosensitivity of cells proficient or deficient in either Ku80 or the DNA-PK catalytic subunit (DNA-PKcs) was investigated. In cells of two Ku80-deficient and two DNA-PKcs-deficient and double-strand break-deficient cell lines, the extent of radiosensitization by heat was not reduced compared to that in both their isogenic gene-complemented counterparts as well as to that in their parental cells. Thus radiosensitization by hyperthermia can be obtained irrespective of the Ku80 or DNA-PKcs status in cells. Therefore, Ku80 or DNA-PKcs and hence nonhomologous DSB end-joining do not play a crucial role in the enhancement of cellular radiosensitivity by hyperthermia.     

Non-homologous end joining requires that the DNA-PK complex undergo an autophosphorylation-dependent rearrangement at DNA ends

Repair of chromosome breaks by non-homologous end joining requires the XRCC4-ligase IV complex, Ku, and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). DNA-PKcs must also retain kinase activity and undergo autophosphorylation at six closely linked sites (ABCDE sites). We describe here an end-joining assay using only purified components that reflects cellular requirements for both Ku and kinase-active DNA-PKcs and investigate the mechanistic basis for these requirements. A need for DNA-PKcs autophosphorylation is sufficient to explain the requirement for kinase activity, in part because autophosphorylation is generally required for end-joining factors to access DNA ends. However, DNA-PKcs with all six ABCDE autophosphorylation sites mutated to alanine allows access to ends through autophosphorylation of other sites, yet our in vitro end-joining assay still reflects the defectiveness of this mutant in cellular end joining. In contrast, mutation of ABCDE sites to aspartate, a phosphorylation mimic, supports high levels of end joining that is now independent of kinase activity. This is likely because DNA-PKcs with aspartate substitutions at ABCDE sites allow access to DNA ends while retaining affinity for Ku-bound ends and stabilizing recruitment of the XRCC4-ligase IV complex. Autophosphorylation at ABCDE sites thus apparently directs a rearrangement of the DNA-PK complex that ensures access to broken ends and joining steps are coupled together within a synaptic complex, making repair more accurate.     

DNA-dependent conformational changes in the Ku heterodimer

Ionizing radiation induces DNA double-strand breaks which are repaired by the nonhomologous end joining (NHEJ) pathway. NHEJ is initiated upon Ku binding to the DNA ends and facilitating an interaction with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). This heterotrimeric DNA-PK complex is then active as a serine/threonine protein kinase. The molecular mechanisms involved in DNA-PK activation are unknown. Considering the crucial role of Ku in this process, we therefore determined the influence of DNA binding on the structure of the Ku heterodimer. Chemical modification with NHS-biotin and mass spectrometry were used to identify sites of modification. Biotinylation of free Ku revealed several reactive lysines on Ku70 and Ku80 which were reduced or eliminated upon DNA binding. Interestingly, in the predicted C-terminal SAP domain of Ku70, biotinylation patterns were observed which suggest a structural change in this region of the protein induced by DNA binding. Limited proteolytic digests of free and DNA-bound Ku revealed a series of unique peptides, again, indicative of a change in the accessibility of the Ku70 and Ku80 C-terminal domains. A 10 kDa peptide was also identified which was preferentially generated under non-DNA-bound conditions and mapped to the C-terminus of Ku70. These results indicate a DNA-dependent movement or structural change in the C-terminal domains of Ku70 and Ku80 that may contribute to DNA-PKcs binding and activation. These results represent the first demonstration of DNA-induced changes in Ku structure and provide a framework for analysis of DNA-PKcs and the mechanism of DNA-PK activation.     

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.     

DNA-PK-dependent phosphorylation of Ku70/80 is not required for non-homologous end joining

The Ku70/80 heterodimer is a major player in non-homologous end joining and the repair of DNA double-strand breaks. Studies suggest that once bound to a DNA double-strand break, Ku recruits the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) to form the DNA-dependent protein kinase holoenzyme complex (DNA-PK). We previously identified four DNA-PK phosphorylation sites on the Ku70/80 heterodimer: serine 6 of Ku70, serine 577 and 580 and threonine 715 of Ku80. This raised the interesting possibility that DNA-PK-dependent phosphorylation of Ku could provide a mechanism for the regulation of non-homologous end joining. Here, using mass spectrometry and phosphospecific antibodies we confirm that these sites are phosphorylated in vitro by purified DNA-PK. However, we show that neither DNA-PK nor the related protein kinase ataxia-telangiectasia mutated (ATM) is required for phosphorylation of Ku at these sites in vivo. Furthermore, Ku containing serine/threonine to alanine mutations at these sites was fully able to complement the radiation sensitivity of Ku negative mammalian cells indicating that phosphorylation at these sites is not required for non-homologous end joining. Interestingly, both Ku70 and Ku80 were phosphorylated in cells treated with the protein phosphatase inhibitor okadaic acid under conditions known to inactivate protein phosphatase 2A-like protein phosphatases. Moreover, okadaic acid-induced phosphorylation of Ku80 was inhibited by nanomolar concentrations of the protein kinase inhibitor staurosporine. These results suggest that the phosphorylation of Ku70 and Ku80 is regulated by a protein phosphatase 2A-like protein phosphatase and a staurosporine sensitive protein kinase in vivo, but that DNA-PK-mediated phosphorylation of Ku is not required for DNA double-strand break repair.     

Autophosphorylation of the catalytic subunit of the DNA-dependent protein kinase is required for efficient end processing during DNA double-strand break repair

The DNA-dependent protein kinase (DNA-PK) plays an essential role in nonhomologous DNA end joining (NHEJ) by initially recognizing and binding to DNA breaks. We have shown that in vitro, purified DNA-PK undergoes autophosphorylation, resulting in loss of activity and disassembly of the kinase complex. Thus, we have suggested that autophosphorylation of the DNA-PK catalytic subunit (DNA-PKcs) may be critical for subsequent steps in DNA repair. Recently, we defined seven autophosphorylation sites within DNA-PKcs. Six of these are tightly clustered within 38 residues of the 4,127-residue protein. Here, we show that while phosphorylation at any single site within the major cluster is not critical for DNA-PK's function in vivo, mutation of several sites abolishes the ability of DNA-PK to function in NHEJ. This is not due to general defects in DNA-PK activity, as studies of the mutant protein indicate that its kinase activity and ability to form a complex with DNA-bound Ku remain largely unchanged. However, analysis of rare coding joints and ends demonstrates that nucleolytic end processing is dramatically reduced in joints mediated by the mutant DNA-PKcs. We therefore suggest that autophosphorylation within the major cluster mediates a conformational change in the DNA-PK complex that is critical for DNA end processing. However, autophosphorylation at these sites may not be sufficient for kinase disassembly.     

DNA-PKcs-dependent signaling of DNA damage in Dictyostelium discoideum

DNA double-strand breaks (DSBs) can be repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). In vertebrates, the first step in NHEJ is recruitment of the DNA-dependent protein kinase (DNA-PK) to DNA termini. DNA-PK consists of a catalytic subunit (DNA-PKcs) that is recruited to DNA ends by the Ku70/Ku80 heterodimer. Although Ku has been identified in a wide variety of organisms, to date DNA-PKcs has only been identified experimentally in vertebrates. Here, we report the identification of DNA-PK in the nonvertebrate Dictyostelium. Dictyostelium Ku80 contains a conserved domain previously implicated in recruiting DNA-PKcs to DNA and consistent with this observation, we have identified DNA-PKcs in the Dictyostelium genome. Disruption of the gene encoding Dictyostelium DNA-PKcs results in sensitivity to DNA DSBs and defective H2AX phosphorylation in response to this form of DNA damage. However, these phenotypes are only apparent when DNA damage is administered in G(1) phase of the cell cycle. These data illustrate a cell cycle-dependent requirement for Dictyostelium DNA-PK in signaling and combating DNA DSBs and represent the first experimental verification of DNA-PKcs in a nonvertebrate organism.     

Binding of inositol hexakisphosphate (IP6) to Ku but not to DNA-PKcs

The nonhomologous DNA end joining (NHEJ) pathway is responsible for repairing a major fraction of double strand DNA breaks in somatic cells of all multicellular eukaryotes. As an indispensable protein in the NHEJ pathway, Ku has been hypothesized to be the first protein to bind at the DNA ends generated at a double strand break being repaired by this pathway. When bound to a DNA end, Ku improves the affinity of another DNA end-binding protein, DNA-PK(cs), to that end. The Ku.DNA-PK(cs) complex is often termed the DNA-PK holoenzyme. It was recently shown that myo-inositol hexakisphosphate (IP(6)) stimulates the joining of complementary DNA ends in a cell free system. Moreover, the binding data suggested that IP(6) bound to DNA-PK(cs) (not to Ku). Here we clearly show that, in fact, IP(6) associates not with DNA-PK(cs), but rather with Ku. Furthermore, the binding of DNA ends and IP(6) to Ku are independent of each other. The possible relationship between inositol phosphate metabolism and DNA repair is discussed in light of these findings.     

Retroviral DNA integration and the DNA damage response

Retroviral DNA integration creates a discontinuity in the host cell chromatin and repair of this damage is required to complete the integration process. As integration and repair are essential for both viral replication and cell survival, it is possible that specific interactions with the host DNA repair systems might provide new cellular targets for human immunodeficiency virus therapy. Various genetic, pharmacological, and biochemical studies have provided strong evidence that postintegration DNA repair depends on components of the nonhomologous end-joining (NHEJ) pathway (DNA-PK (DNA-dependent protein kinase), Ku, Xrcc4, DNA ligase IV) and DNA damage-sensing pathways (Atr (Atm and Rad related), gamma-H2AX). Furthermore, deficiencies in NHEJ components result in susceptibility to apoptotic cell death following retroviral infection. Here, we review these findings and discuss other ways that retroviral DNA intermediates may interact with the host DNA damage signaling and repair pathways.