Distinct roles for DNA-PK,ATM and ATR in RPA phosphorylation and checkpoint activation in response to replication stress

DNA damage encountered by DNA replication forks poses risks of genome destabilization, a precursor to carcinogenesis. Damage checkpoint systems cause cell cycle arrest, promote repair and induce programed cell death when damage is severe. Checkpoints are critical parts of the DNA damage response network that act to suppress cancer. DNA damage and perturbation of replication machinery causes replication stress, characterized by accumulation of single-stranded DNA bound by replication protein A (RPA), which triggers activation of ataxia telangiectasia and Rad3 related (ATR) and phosphorylation of the RPA32, subunit of RPA, leading to Chk1 activation and arrest. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) [a kinase related to ataxia telangiectasia mutated (ATM) and ATR] has well characterized roles in DNA double-strand break repair, but poorly understood roles in replication stress-induced RPA phosphorylation. We show that DNA-PKcs mutant cells fail to arrest replication following stress, and mutations in RPA32 phosphorylation sites targeted by DNA-PKcs increase the proportion of cells in mitosis, impair ATR signaling to Chk1 and confer a G2/M arrest defect. Inhibition of ATR and DNA-PK (but not ATM), mimic the defects observed in cells expressing mutant RPA32. Cells expressing mutant RPA32 or DNA-PKcs show sustained H2AX phosphorylation in response to replication stress that persists in cells entering mitosis, indicating inappropriate mitotic entry with unrepaired damage.     

DNA structure-specific priming of ATR activation by DNA-PKcs

Three phosphatidylinositol-3-kinase–related protein kinases implement cellular responses to DNA damage. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia-telangiectasia mutated respond primarily to DNA double-strand breaks (DSBs). Ataxia-telangiectasia and RAD3-related (ATR) signals the accumulation of replication protein A (RPA)–covered single-stranded DNA (ssDNA), which is caused by replication obstacles. Stalled replication intermediates can further degenerate and yield replication-associated DSBs. In this paper, we show that the juxtaposition of a double-stranded DNA end and a short ssDNA gap triggered robust activation of endogenous ATR and Chk1 in human cell-free extracts. This DNA damage signal depended on DNA-PKcs and ATR, which congregated onto gapped linear duplex DNA. DNA-PKcs primed ATR/Chk1 activation through DNA structure-specific phosphorylation of RPA32 and TopBP1. The synergistic activation of DNA-PKcs and ATR suggests that the two kinases combine to mount a prompt and specific response to replication-born DSBs.     

DNA-PK phosphorylation of RPA32 Ser4/Ser8 regulates replication stress checkpoint activation,fork restart,homologous recombination and mitotic catastrophe

Genotoxins and other factors cause replication stress that activate the DNA damage response (DDR), comprising checkpoint and repair systems. The DDR suppresses cancer by promoting genome stability, and it regulates tumor resistance to chemo- and radiotherapy. Three members of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, ATM, ATR, and DNA-PK, are important DDR proteins. A key PIKK target is replication protein A (RPA), which binds single-stranded DNA and functions in DNA replication, DNA repair, and checkpoint signaling. An early response to replication stress is ATR activation, which occurs when RPA accumulates on ssDNA. Activated ATR phosphorylates many targets, including the RPA32 subunit of RPA, leading to Chk1 activation and replication arrest. DNA-PK also phosphorylates RPA32 in response to replication stress, and we demonstrate that cells with DNA-PK defects, or lacking RPA32 Ser4/Ser8 targeted by DNA-PK, confer similar phenotypes, including defective replication checkpoint arrest, hyper-recombination, premature replication fork restart, failure to block late origin firing, and increased mitotic catastrophe. We present evidence that hyper-recombination in these mutants is ATM-dependent, but the other defects are ATM-independent. These results indicate that DNA-PK and ATR signaling through RPA32 plays a critical role in promoting genome stability and cell survival in response to replication stress.     

Proteasome inhibition suppresses DNA-dependent protein kinase activation caused by camptothecin

The ubiquitin–proteasome pathway plays an important role in DNA damage signaling and repair by facilitating the recruitment and activation of DNA repair factors and signaling proteins at sites of damaged chromatin. Proteasome activity is generally not thought to be required for activation of apical signaling kinases including the PI3K-related kinases (PIKKs) ATM, ATR, and DNA-PK that orchestrate downstream signaling cascades in response to diverse genotoxic stimuli. In a previous work, we showed that inhibition of the proteasome by MG-132 suppressed 53BP1 (p53 binding protein1) phosphorylation as well as RPA2 (replication protein A2) phosphorylation in response to the topoisomerase I (TopI) poison camptothecin (CPT). To address the mechanism of proteasome-dependent RPA2 phosphorylation, we investigated the effects of proteasome inhibitors on the upstream PIKKs. MG-132 sharply suppressed CPT-induced DNA-PKcs autophosphorylation, a marker of the activation, whereas the phosphorylation of ATM and ATR substrates was only slightly suppressed by MG-132, suggesting that DNA-PK among the PIKKs is specifically regulated by the proteasome in response to CPT. On the other hand, MG-132 did not suppress DNA-PK activation in response to UV or IR. MG-132 blocked the interaction between DNA-PKcs and Ku heterodimer enhanced by CPT, and hydroxyurea pre-treatment completely abolished CPT-induced DNA-PKcs autophosphorylation, indicating a requirement for ongoing DNA replication. CPT-induced TopI degradation occurred independent of DNA-PK activation, suggesting that DNA-PK activation does not require degradation of trapped TopI complexes. The combined results suggest that CPT-dependent replication fork collapse activates DNA-PK signaling through a proteasome dependent, TopI degradation-independent pathway. The implications of DNA-PK activation in the context of TopI poison-based therapies are discussed.     

Selective inhibition of the DNA-dependent protein kinase (DNA-PK) by the radiosensitizing agent caffeine

Caffeine inhibits cell cycle checkpoints, sensitizes cells to ionizing radiation-induced cell killing and inhibits the protein kinase activity of two cell cycle checkpoint regulators, Ataxia-Telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR). In contrast, caffeine has been reported to have little effect on the protein kinase activity of the DNA-dependent protein kinase (DNA-PK), which is essential for the repair of DNA double-strand breaks. Previously, we reported that DNA-PK phosphorylates Thr²¹ of the 32 kDa subunit of replication protein A (RPA32) in response to camptothecin. In this report we demonstrate that the camptothecin-induced phosphorylation of RPA32 on Thr²¹ is inhibited by 2 mM caffeine. In addition, we show that caffeine inhibits immunoprecipitated and purified DNA-PK, as well as DNA-PK in cell extracts, with an IC₅₀ of 0.2–0.6 mM. Caffeine inhibited DNA-PK activity through a mixed non-competitive mechanism with respect to ATP. In contrast, 10-fold higher concentrations of caffeine were required to inhibit DNA-PK autophosphorylation in vitro and caffeine failed to inhibit DNA-PKcs dependent double-strand break repair in vivo. These data suggest that while DNA-PK does not appear to be the target of caffeine-induced radiosensitization, caffeine cannot be used to differentiate between ATM, ATR and DNA- PK-dependent substrate phosphorylation in vivo.     

Phosphatidyl inositol 3-kinase-like serine/threonine protein kinases (PIKKs) are required for DNA damage-induced phosphorylation of the 32 kDa subunit of replication protein A at threonine 21

Replication protein A (RPA) is a single-stranded DNA (ssDNA) binding protein involved in various processes, including nucleotide excision repair and DNA replication. The 32 kDa subunit of RPA (RPA32) is phosphorylated in response to various DNA-damaging agents, and two protein kinases, ataxia-telangiectasia mutated (ATM) and the DNA-dependent protein kinase (DNA-PK) have been implicated in DNA damage-induced phosphorylation of RPA32. However, the relative roles of ATM and DNA-PK in the site-specific DNA damage-induced phosphorylation of RPA32 have not been reported. Here we generated a phosphospecific antibody that recognizes Thr²¹-phosphorylated RPA32. We show that both DNA-PK and ATM phosphorylate RPA32 on Thr²¹ in vitro. Ionizing radiation (IR)-induced phosphorylation of RPA32 on Thr²¹ was defective in ATM-deficient cells, while camptothecin (CPT)-induced phosphorylation of RPA32 on Thr²¹ was defective in cells lacking functional DNA-PK. Neither ATM nor DNA-PK was required for etoposide (ETOP)-induced RPA32 Thr²¹ phosphorylation. However, two inhibitors of the ATM- and Rad3-related (ATR) protein kinase activity prevented ETOP-induced Thr²¹ phosphorylation. Inhibition of DNA replication prevented both the IR- and CPT-induced phosphorylation of Thr²¹, whereas ETOP-induced Thr²¹ phosphorylation did not require active DNA replication. Thus, the regulation of RPA32 Thr²¹ phosphorylation by multiple DNA damage response protein kinases suggests that Thr²¹ phosphorylation of RPA32 is a crucial step within the DNA damage response.     

Involvement of DNA-dependent protein kinase in regulation of stress-induced JNK activation.

DNA-dependent protein kinase (DNA-PK) is composed of a 460-kDa catalytic subunit and the regulatory subunits Ku70 and Ku80. The complex is activated on DNA damage and plays an essential role in double-strand-break repair and V(D)J recombination. In addition, DNA-PK is involved in S-phase checkpoint arrest following irradiation, although its role in damage-induced checkpoint arrest is not clear. In an effort to understand the role of DNA-PK in damage signaling, human and mouse cells containing the DNA-PK catalytic subunit (DNA-PKcs proficient) were compared with those lacking DNA-PKcs for c-Jun N-terminal kinase (JNK) activity that mediates physiologic responses to DNA damage. The DNA-PKcs-proficient cells showed much tighter regulation of JNK activity after DNA damage, while the level of JNK protein in both cell lines remained unchanged. The JNK proteins physically associated with DNA-PKcs and Ku70/Ku80 heterodimer, and the interaction was significantly stimulated after DNA damage. Various JNK isoforms not only contained a DNA-PK phosphorylation consensus site (serine followed by glutamine) but also were phosphorylated by DNA-PK in vitro. Together, our results suggest that DNA damage induces physical interaction between DNA-PK and JNK, which may in turn negatively affect JNK activity through JNK phosphorylation by DNA-PK.     

UV-induced RPA phosphorylation is increased in the absence of DNA polymerase eta and requires DNA-PK

Signaling from arrested replication forks plays a role in maintaining genome stability. We have investigated this process in xeroderma pigmentosum variant cells that carry a mutation in the POLH gene and lack functional DNA polymerase eta (poleta). Poleta is required for error-free bypass of UV-induced cyclobutane pyrimidine dimers; in the absence of poleta in XPV cells, DNA replication is arrested at sites of UV-induced DNA damage, and mutagenic bypass of lesions is ultimately carried out by other, error-prone, DNA polymerases. The present study investigates whether poleta expression influences the activation of a number of UV-induced DNA damage responses. In a stably transfected XPV cell line (TR30-9) in which active poleta can be induced by addition of tetracycline, expression of poleta determines the extent of DNA double-strand break formation following UV-irradiation. UV-induced phosphorylation of replication protein A (RPA), a key DNA-binding protein involved in DNA replication, repair and recombination, is increased in cells lacking poleta compared to when poleta is expressed in the same cell line. To identify the protein kinase responsible for increased UV-induced hyperphosphorylation of the p34 subunit of RPA, we have used NU7441, a specific small molecule inhibitor of DNA-PK. DNA-PK is necessary for RPA p34 hyperphosphorylation, but DNA-PK-mediated phosphorylation is not required for recruitment of RPA p34 into nuclear foci in response to UV-irradiation. The results demonstrate that activation of a UV-induced DNA damage response pathway, involving phosphorylation of RPA p34 by DNA-PK, is enhanced in cells lacking poleta.     

Sequential and synergistic modification of human RPA stimulates chromosomal DNA repair

The activity of human replication protein A (RPA) in DNA replication and repair is regulated by phosphorylation of the middle RPA2 subunit. It has previously been shown that up to nine different N-terminal residues are modified in vivo and in response to genotoxic stress. Using a novel antibody against phospho-Ser(29), a moiety formed by cyclin-Cdk, we observed that RPA2 was phosphorylated during mitosis in nonstressed cells. Robust phosphorylation of Ser(29) was also seen in interphase cells following treatment with the DNA-damaging agent camptothecin, a rare example of stress stimulating the modification of a repair factor by cyclin-Cdk. RPA2 phosphorylation is regulated both in cis and trans. Cis-phosphorylation follows a preferred pathway. (That is, the initial modification of Ser(33) by ATR stimulates subsequent phosphorylation of Cdk sites Ser(23) and Ser(29)). These events then facilitate modification of Thr(21) and extreme N-terminal sites Ser(4) and Ser(8), probably by DNA-PK. Our data also indicate that the phosphorylation of one RPA molecule can influence the phosphorylation of other RPA molecules in trans. Cells in which endogenous RPA2 was "replaced" with a double S23A/S29A-RPA2 mutant were seen to have an abnormal cell cycle distribution both in normal and in stressed cells. Such cells also showed aberrant DNA damage-dependent RPA foci and had persistent staining of gammaH2AX following DNA damage. Our data indicate that RPA phosphorylation facilitates chromosomal DNA repair. We postulate that the RPA phosphorylation pattern provides a means to regulate the DNA repair pathway utilized.     

Replication Protein A (RPA): The Eukaryotic SSB

Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein that is highly conserved in eukaryotes. RPA plays essential roles in many aspects of nucleic acid metabolism, including DNA replication, nucleotide excision repair, and homologous recombination. In this review, we provide a comprehensive overview of RPA structure and function and highlight the more recent developments in these areas. The last few years have seen major advances in our understanding of the mechanism of RPA binding to DNA, including the structural characterization of the primary DNA-binding domains (DBD) and the identification of two secondary DBDs. Moreover, evidence indicates that RPA utilizes a multistep pathway to bind single-stranded DNA involving a particular molecular polarity of RPA, a mechanism that is apparently used to facilitate origin denaturation. In addition to its mechanistic roles, RPA interacts with many key factors in nucleic acid metabolism, and we discuss the critical nature of many of these interactions to DNA metabolism. RPA is a phosphorylation target for DNA-dependent protein kinase (DNA-PK) and likely the ataxia telangiectasia-mutated gene (ATM) protein kinase, and recent observations are described that suggest that RPA phosphorylation plays a significant modulatory role in the cellular response to DNA damage.     

HARP preferentially co-purifies with RPA bound to DNA-PK and blocks RPA phosphorylation

The HepA-related protein (HARP/SMARCAL1) is an ATP-dependent annealing helicase that is capable of rewinding DNA structures that are stably unwound due to binding of the single-stranded DNA (ssDNA)-binding protein Replication Protein A (RPA). HARP has been implicated in maintaining genome integrity through its role in DNA replication and repair, two processes that generate RPA-coated ssDNA. In addition, mutations in HARP cause a rare disease known as Schimke immuno-osseous dysplasia. In this study, we purified HARP containing complexes with the goal of identifying the predominant factors that stably associate with HARP. We found that HARP preferentially interacts with RPA molecules that are bound to the DNA-dependent protein kinase (DNA-PK). We also found that RPA is phosphorylated by DNA-PK in vitro, while the RPA-HARP complexes are not. Our results suggest that, in addition to its annealing helicase activity, which eliminates the natural binding substrate for RPA, HARP blocks the phosphorylation of RPA by DNA-PK.     

HARP preferentially co-purifies with RPA bound to DNA-PK and blocks RPA phosphorylation

The HepA-related protein (HARP/SMARCAL1) is an ATP-dependent annealing helicase that is capable of rewinding DNA structures that are stably unwound due to binding of the single-stranded DNA (ssDNA)-binding protein Replication Protein A (RPA). HARP has been implicated in maintaining genome integrity through its role in DNA replication and repair, two processes that generate RPA-coated ssDNA. In addition, mutations in HARP cause a rare disease known as Schimke immuno-osseous dysplasia. In this study, we purified HARP containing complexes with the goal of identifying the predominant factors that stably associate with HARP. We found that HARP preferentially interacts with RPA molecules that are bound to the DNA-dependent protein kinase (DNA-PK). We also found that RPA is phosphorylated by DNA-PK in vitro, while the RPA-HARP complexes are not. Our results suggest that, in addition to its annealing helicase activity, which eliminates the natural binding substrate for RPA, HARP blocks the phosphorylation of RPA by DNA-PK.     

Artemis phosphorylated by DNA-dependent protein kinase associates preferentially with discrete regions of chromatin

Artemis is a nuclear phosphoprotein required for genomic integrity whose phosphorylation is increased subsequent to DNA damage. Artemis phosphorylation by the DNA-dependent protein kinase (DNA-PK) and the association of Artemis with DNA-PK catalytic subunit (DNA-PKcs) have been proposed to be crucial for the variable, diversity, joining (V(D)J) reaction, genomic stability and cell survival in response to double-stranded DNA breaks. The exact nature of the effectors of Artemis phosphorylation is presently being debated. Here, we have delimited the interface on Artemis required for its association with DNA-PKcs and present the characterization of six DNA-PK phosphorylation sites on Artemis whose phosphorylation shows dependence on its association with DNA-PKcs and is induced by double-stranded DNA damage. Surprisingly, DNA-PKcs Artemis association appeared to be dispensable in a V(D)J recombination assay with stably integrated DNA substrates. Phosphorylation at two of the sites on Artemis, S516 and S645, was verified in vivo using phosphospecific antibodies. Basal Artemis S516 and S645 phosphorylation in vivo showed a significant dependence on DNA-PKcs association. However, regardless of its association with DNA-PKcs, phosphorylation of Artemis at both S516 and S645 was stimulated in response to the double-stranded DNA-damaging agent bleomycin, albeit to a lesser extent. This suggests that additional factors contribute to promote DNA damage-induced Artemis phosphorylation. Intriguingly, pS516/pS645 Artemis was concentrated in chromatin-associated nuclear foci in na?ve cells. These foci were maintained upon DNA damage but failed to overlap with the damage-induced gammaH2AX. These results provide the expectation of a specific role for DNA-PK-phosphorylated Artemis in both na?ve and damaged cells.     

DNA-PK-dependent RPA2 hyperphosphorylation facilitates DNA repair and suppresses sister chromatid exchange

Hyperphosphorylation of RPA2 at serine 4 and serine 8 (S4, S8) has been used as a marker for activation of the DNA damage response. What types of DNA lesions cause RPA2 hyperphosphorylation, which kinase(s) are responsible for them, and what is the biological outcome of these phosphorylations, however, have not been fully investigated. In this study we demonstrate that RPA2 hyperphosphorylation occurs primarily in response to genotoxic stresses that cause high levels of DNA double-strand breaks (DSBs) and that the DNA-dependent protein kinase complex (DNA-PK) is responsible for the modifications in vivo. Alteration of S4, S8 of RPA2 to alanines, which prevent phosphorylations at these sites, caused increased mitotic entry with concomitant increases in RAD51 foci and homologous recombination. Taken together, our results demonstrate that RPA2 hyperphosphorylation by DNA-PK in response to DSBs blocks unscheduled homologous recombination and delays mitotic entry. This pathway thus permits cells to repair DNA damage properly and increase cell viability.     

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.     

DNA Double-Strand Break Formation upon UV-Induced Replication Stress Activates ATM and DNA-PKcs Kinases

The phosphatidylinositol 3-kinase-like protein kinases, including ATM (ataxia-telangiectasia mutated), ATR (ataxia-telangiectasia and Rad3 related), and DNA-PKcs (DNA-dependent protein kinase catalytic subunit), are the main kinases activated following various assaults on DNA. Although ATM and DNA-PKcs kinases are activated upon DNA double-strand breaks, evidence suggests that these kinases are rapidly phosphorylated by ATR kinase upon UV irradiation; thus, these kinases may also participate in the response to replication stress. Using UV-induced replication stress, we further characterize whether ATM and DNA-PKcs kinase activities are also involved in the cellular response. Contrary to the rapid activation of the ATR-dependent pathway, ATM-dependent Chk2 and KAP-1 phosphorylations, as well as DNA-PKcs Ser2056 autophosphorylation, reach their peak level at 4 to 8 h after UV irradiation. The delayed kinetics of ATM- and DNA-PKcs-dependent phosphorylations also correlated with a surge in H2AX phosphorylation, suggesting that double-strand break formation resulting from collapse of replication forks is responsible for the activation of ATM and DNA-PKcs kinases. In addition, we observed that some phosphorylation events initiated by ATR kinase in the response to UV were mediated by ATM at a later phase of the response. Furthermore, the S-phase checkpoint after UV irradiation was defective in ATM-deficient cells. These results suggest that the late increase of ATM activity is needed to complement the decreasing ATR activity for maintaining a vigilant checkpoint regulation upon replication stress.     

Phosphorylation of human replication protein A by the DNA-dependent protein kinase is involved in the modulation of DNA replication.

The single-stranded DNA-binding protein, Replication Protein A (RPA), is a heterotrimeric complex with subunits of 70, 32 and 14 kDa involved in DNA metabolism. RPA may be a target for cellular regulation; the 32 kDa subunit (RPA32) is phosphorylated by several cellular kinases including the DNA-dependent protein kinase (DNA-PK). We have purified a mutant hRPA complex lacking amino acids 1-33 of RPA32 (rhRPA x 32delta1-33). This mutant bound ssDNA and supported DNA replication; however, rhRPA x 32delta1-33 was not phosphorylated under replication conditions or directly by DNA-PK. Proteolytic mapping revealed that all the sites phosphorylated by DNA-PK are contained on residues 1-33 of RPA32. When wild-type RPA was treated with DNA-PK and the mixture added to SV40 replication assays, DNA replication was supported. In contrast, when rhRPA x 32delta1-33 was treated with DNA-PK, DNA replication was strongly inhibited. Because untreated rhRPA x 32delta1-33 is fully functional, this suggests that the N-terminus of RPA is needed to overcome inhibitory effects of DNA-PK on other components of the DNA replication system. Thus, phosphorylation of RPA may modulate DNA replication indirectly, through interactions with other proteins whose activity is modulated by phosphorylation.     

Enhanced DNA-PK-mediated RPA2 hyperphosphorylation in DNA polymerase eta-deficient human cells treated with cisplatin and oxaliplatin

The chemotherapeutic drugs cisplatin and oxaliplatin act by induction of DNA damage, including monoadducts, intrastrand and interstrand crosslinks. An increased understanding of the repair and replication of platinum-damaged DNA is required to improve the effectiveness of these drugs in killing cancer cells. We have investigated the effect of expression of DNA polymerase eta (poleta), a translesion synthesis (TLS) enzyme, on the response of human cell lines to cisplatin and oxaliplatin. Poleta-deficient cells are more sensitive to both drugs than are normal cells. In poleta-deficient cells, drug treatment leads to prolonged S-phase arrest, and increased phosphorylation of the phosphatidylinositol-3-kinase-related protein kinase (PIKK) substrates Chk1, p95/Nbs1 and RPA2, the 34kDa subunit of replication protein A. Cisplatin- and oxaliplatin-induced hyperphosphorylation of RPA2, and association of the hyperphosphorylated protein with chromatin, is elevated in poleta-deficient cells. Cisplatin-induced phosphorylation of RPA2 on serine 4/serine 8, but not on serine 33, is inhibited by the DNA-PK inhibitor, NU7441, but not by the ATM inhibitor, KU-55933. Cisplatin-induced DNA-PK-dependent hyperphosphorylation of RPA2 on serine 4/serine 8 occurs after recruitment of RPA to chromatin, as determined by immunofluorescence and by subcellular fractionation. ATR is required both for recruitment of RPA2 to chromatin and its subsequent hyperphosphorylation on serine 4/serine 8 by DNA-PK, since CGK733, an inhibitor of ATM and ATR, blocked both recruitment and hyperphosphorylation. Thus, increased sensitivity to cisplatin and oxaliplatin in DNA poleta-deficient cells is associated with prolonged S-phase arrest, and enhanced PIKK-signalling, in particular activation of DNA-PK-dependent hyperphosphorylation of RPA2 on serines 4 and 8.     

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.