Deciphering the role of distinct DNA-PK phosphorylations at collapsed replication forks.

It has recently been established that the marked sensitivity of ATM deficient cells to topoisomerase poisons like camptothecin (Cpt) results from unrestrained end-joining of DNA ends at collapsed replication forks that is mediated by the non-homologous end joining [NHEJ] pathway and results in the induction of copious numbers of genomic alterations, termed "toxic NHEJ". Ablation of core components of the NHEJ pathway reverses the Cpt sensitivity of ATM deficient cells, but inhibition of DNA-PKcs does not. Here, we show that complete ablation of DNA-PKcs partially reverses the Cpt sensitivity of ATM deficient cells; thus, ATM deficient cells lacking DNA-PKcs are more resistant to Cpt than cells expressing DNA-PKcs. However, the relative sensitivity of DNA-PKcs proficient ATM deficient cells is inversely proportional to DNA-PKcs expression levels. These data suggest that DNA-PK may phosphorylate an ATM target (that contributes to Cpt resistance), explaining partial rescue of Cpt sensitivity in cells expressing high levels of DNA-PKcs. Although crippling NHEJ function by mutagenic blockade of the critical ABCDE autophosphorylation sites in DNA-PKcs also sensitizes cells to Cpt, this sensitization apparently occurs by a distinct mechanism from ATM ablation because blockade of these sites actually rescues ATM deficient cells from toxic NHEJ. These data are consistent with autophosphorylation of the ABCDE sites (and not ATM mediated phosphorylation) in response to Cpt-induced damage. In contrast, blockade of S3205 (an ATM dependent phosphorylation site in DNA-PKcs) that minimally impacts NHEJ, increases Cpt sensitivity. In sum, these data suggest that ATM and DNA-PK cooperate to facilitate Cpt-induced DNA damage, and that ATM phosphorylation of S3205 facilitates appropriate repair at collapsed replication forks.

Deciphering phenotypic variance in different models of DNA-PKcs deficiency.

DNA-PKcs deficiency has been studied in numerous animal models and cell culture systems. In previous studies of kinase inactivating mutations in cell culture systems, ablation of DNA-PK's catalytic activity results in a cell phenotype that is virtually indistinguishable from that ascribed to complete loss of the enzyme. However, a recent compelling study demonstrates a remarkably more severe phenotype in mice harboring a targeted disruption of DNA-PK's ATP binding site as compared to DNA-PKcs deficient mice. Here we investigate the mechanism for these divergent results. We find that kinase inactivating DNA-PKcs mutants markedly radiosensitize immortalized DNA-PKcs deficient cells, but have no substantial effects on transformed DNA-PKcs deficient cells. Since the non-homologous end joining mechanism likely functions similarly in all of these cell strains, it seems unlikely that kinase inactive DNA-PK could impair the end joining mechanism in some cell types, but not in others. In fact, we observed no significant differences in either episomal or chromosomal end joining assays in cells expressing kinase inactivated DNA-PKcs versus no DNA-PKcs. Several potential explanations could explain these data including a non-catalytic role for DNA-PKcs in promoting cell death, or alteration of gene expression by loss of DNA-PKcs as opposed to inhibition of its catalytic activity. Finally, controversy exists as to whether DNA-PKcs autophosphorylates or is the target of other PIKKs; we present data demonstrating that DNA-PK primarily autophosphorylates.

The ATM Kinase Restrains Joining of Both VDJ Signal and Coding Ends.

The evidence that ATM affects resolution of RAG-induced DNA double-strand breaks is profuse and unequivocal; moreover, it is clear that the RAG complex itself cooperates (in an undetermined way) with ATM to facilitate repair of these double-strand breaks by the classical nonhomologous end-joining pathway. The mechanistic basis for the cooperation between ATM and the RAG complex has not been defined, although proposed models invoke ATM and RAG2's C terminus in maintaining the RAG postcleavage complex. In this study, we show that ATM reduces the rate of both coding and signal joining in a robust episomal assay; we suggest that this is the result of increased stability of the postcleavage complex. ATM's ability to inhibit VDJ joining requires its enzymatic activity. The noncore C termini of both RAG1 and RAG2 are also required for ATM's capacity to limit signal (but not coding) joining. Moreover, potential phosphorylation targets within the C terminus of RAG2 are also required for ATM's capacity to limit signal joining. These data suggest a model whereby the RAG signal end complex is stabilized by phosphorylation of RAG2 by ATM.

Restoration of ATM Expression in DNA-PKcs-Deficient Cells Inhibits Signal End Joining.

Unlike most DNA-dependent protein kinase, catalytic subunit (DNA-PKcs)-deficient mouse cell strains, we show in the present study that targeted deletion of DNA-PKcs in two different human cell lines abrogates VDJ signal end joining in episomal assays. Although the mechanism is not well defined, DNA-PKcs deficiency results in spontaneous reduction of ATM expression in many cultured cell lines (including those examined in this study) and in DNA-PKcs-deficient mice. We considered that varying loss of ATM expression might explain differences in signal end joining in different cell strains and animal models, and we investigated the impact of ATM and/or DNA-PKcs loss on VDJ recombination in cultured human and rodent cell strains. To our surprise, in DNA-PKcs-deficient mouse cell strains that are proficient in signal end joining, restoration of ATM expression markedly inhibits signal end joining. In contrast, in DNA-PKcs-deficient cells that are deficient in signal end joining, complete loss of ATM enhances signal (but not coding) joint formation. We propose that ATM facilitates restriction of signal ends to the classical nonhomologous end-joining pathway.

DNA-PKcs and PARP1 Bind to Unresected Stalled DNA Replication Forks Where They Recruit XRCC1 to Mediate Repair.

A series of critical pathways are responsible for the detection, signaling, and restart of replication forks that encounter blocks during S-phase progression. Small base lesions may obstruct replication fork progression and processing, but the link between repair of small lesions and replication forks is unclear. In this study, we investigated a hypothesized role for DNA-PK, an important enzyme in DNA repair, in cellular responses to DNA replication stress. The enzyme catalytic subunit DNA-PKcs was phosphorylated on S2056 at sites of stalled replication forks in response to short hydroxyurea treatment. Using DNA fiber experiments, we found that catalytically active DNA-PK was required for efficient replication restart of stalled forks. Furthermore, enzymatically active DNA-PK was also required for PARP-dependent recruitment of XRCC1 to stalled replication forks. This activity was enhanced by preventing Mre11-dependent DNA end resection, suggesting that XRCC1 must be recruited early to an unresected stalled fork. We also found that XRCC1 was required for effective restart of a subset of stalled replication forks. Overall, our work suggested that DNA-PK and PARP-dependent recruitment of XRCC1 is necessary to effectively protect, repair, and restart stalled replication forks, providing new insight into how genomic stability is preserved.

Polo-like kinase 1 (PLK1) and protein phosphatase 6 (PP6) regulate DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation in mitosis.

The protein kinase activity of the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) and its autophosphorylation are critical for DBS (DNA double-strand break) repair via NHEJ (non-homologous end-joining). Recent studies have shown that depletion or inactivation of DNA-PKcs kinase activity also results in mitotic defects. DNA-PKcs is autophosphorylated on Ser2056, Thr2647 and Thr2609 in mitosis and phosphorylated DNA-PKcs localize to centrosomes, mitotic spindles and the midbody. DNA-PKcs also interacts with PP6 (protein phosphatase 6), and PP6 has been shown to dephosphorylate Aurora A kinase in mitosis. Here we report that DNA-PKcs is phosphorylated on Ser3205 and Thr3950 in mitosis. Phosphorylation of Thr3950 is DNA-PK-dependent, whereas phosphorylation of Ser3205 requires PLK1 (polo-like kinase 1). Moreover, PLK1 phosphorylates DNA-PKcs on Ser3205 in vitro and interacts with DNA-PKcs in mitosis. In addition, PP6 dephosphorylates DNA-PKcs at Ser3205 in mitosis and after IR (ionizing radiation). DNA-PKcs also phosphorylates Chk2 on Thr68 in mitosis and both phosphorylation of Chk2 and autophosphorylation of DNA-PKcs in mitosis occur in the apparent absence of Ku and DNA damage. Our findings provide mechanistic insight into the roles of DNA-PKcs and PP6 in mitosis and suggest that DNA-PKcs' role in mitosis may be mechanistically distinct from its well-established role in NHEJ.

Unraveling the complexities of DNA-dependent protein kinase autophosphorylation.

DNA-dependent protein kinase (DNA-PK) orchestrates DNA repair by regulating access to breaks through autophosphorylations within two clusters of sites (ABCDE and PQR). Blocking ABCDE phosphorylation (by alanine mutation) imparts a dominant negative effect, rendering cells hypersensitive to agents that cause DNA double-strand breaks. Here, a mutational approach is used to address the mechanistic basis of this dominant negative effect. Blocking ABCDE phosphorylation hypersensitizes cells to most types of DNA damage (base damage, cross-links, breaks, and damage induced by replication stress), suggesting that DNA-PK binds DNA ends that result from many DNA lesions and that blocking ABCDE phosphorylation sequesters these DNA ends from other repair pathways. This dominant negative effect requires DNA-PK's catalytic activity, as well as phosphorylation of multiple (non-ABCDE) DNA-PK catalytic subunit (DNA-PKcs) sites. PSIPRED analysis indicates that the ABCDE sites are located in the only contiguous extended region of this huge protein that is predicted to be disordered, suggesting a regulatory role(s) and perhaps explaining the large impact ABCDE phosphorylation has on the enzyme's function. Moreover, additional sites in this disordered region contribute to the ABCDE cluster. These data, coupled with recent structural data, suggest a model whereby early phosphorylations promote initiation of nonhomologous end joining (NHEJ), whereas ABCDE phosphorylations, potentially located in a "hinge" region between the two domains, lead to regulated conformational changes that initially promote NHEJ and eventually disengage NHEJ.

PRKDC mutations in a SCID patient with profound neurological abnormalities.

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs; encoded by PRKDC) functions in DNA non-homologous end-joining (NHEJ), the major DNA double strand break (DSB) rejoining pathway. NHEJ also functions during lymphocyte development, joining V(D)J recombination intermediates during antigen receptor gene assembly. Here, we describe a patient with compound heterozygous mutations in PRKDC, low DNA-PKcs expression, barely detectable DNA-PK kinase activity, and impaired DSB repair. In a heterologous expression system, we found that one of the PRKDC mutations inactivated DNA-PKcs, while the other resulted in dramatically diminished but detectable residual function. The patient suffered SCID with reduced or absent T and B cells, as predicted from PRKDC-deficient animal models. Unexpectedly, the patient was also dysmorphic; showed severe growth failure, microcephaly, and seizures; and had profound, globally impaired neurological function. MRI scans revealed microcephaly-associated cortical and hippocampal dysplasia and progressive atrophy over 2 years of life. These neurological features were markedly more severe than those observed in patients with deficiencies in other NHEJ proteins. Although loss of DNA-PKcs in mice, dogs, and horses was previously shown not to impair neuronal development, our findings demonstrate a stringent requirement for DNA-PKcs during human neuronal development and suggest that high DNA-PK protein expression is required to sustain efficient pre- and postnatal neurogenesis.

XRCC4's interaction with XLF is required for coding (but not signal) end joining.

XRCC4 and XLF are structurally related proteins important for DNA Ligase IV function. XRCC4 forms a tight complex with DNA Ligase IV while XLF interacts directly with XRCC4. Both XRCC4 and XLF form homodimers that can polymerize as heterotypic filaments independently of DNA Ligase IV. Emerging structural and in vitro biochemical data suggest that XRCC4 and XLF together generate a filamentous structure that promotes bridging between DNA molecules. Here, we show that ablating XRCC4's affinity for XLF results in DNA repair deficits including a surprising deficit in VDJ coding, but not signal end joining. These data are consistent with a model whereby XRCC4/XLF complexes hold DNA ends together--stringently required for coding end joining, but dispensable for signal end joining. Finally, DNA-PK phosphorylation of XRCC4/XLF complexes disrupt DNA bridging in vitro, suggesting a regulatory role for DNA-PK's phosphorylation of XRCC4/XLF complexes.

Choosing the right path: does DNA-PK help make the decision?

DNA double-strand breaks are extremely harmful lesions that can lead to genomic instability and cell death if not properly repaired. There are at least three pathways that are responsible for repairing DNA double-strand breaks in mammalian cells: non-homologous end joining, homologous recombination and alternative non-homologous end joining. Here we review each of these three pathways with an emphasis on the role of the DNA-dependent protein kinase, a critical component of the non-homologous end joining pathway, in influencing which pathway is ultimately utilized for repair.

Inhibition of homologous recombination by DNA-dependent protein kinase requires kinase activity, is titratable, and is modulated by autophosphorylation.

How a cell chooses between nonhomologous end joining (NHEJ) and homologous recombination (HR) to repair a double-strand break (DSB) is a central and largely unanswered question. Although there is evidence of competition between HR and NHEJ, because of the DNA-dependent protein kinase (DNA-PK)'s cellular abundance, it seems that there must be more to the repair pathway choice than direct competition. Both a mutational approach and chemical inhibition were utilized to address how DNA-PK affects HR. We find that DNA-PK's ability to repress HR is both titratable and entirely dependent on its enzymatic activity. Still, although requisite, robust enzymatic activity is not sufficient to inhibit HR. Emerging data (including the data presented here) document the functional complexities of DNA-PK's extensive phosphorylations that likely occur on more than 40 sites. Even more, we show here that certain phosphorylations of the DNA-PK large catalytic subunit (DNA-PKcs) clearly promote HR while inhibiting NHEJ, and we conclude that the phosphorylation status of DNA-PK impacts how a cell chooses to repair a DSB.

The role of hRev7, the accessory subunit of hPolζ, in translesion synthesis past DNA damage induced by benzo[a]pyrene diol epoxide (BPDE).

BACKGROUND: DNA polymerase zeta (Polζ) is a specialized DNA polymerase that, unlike classical replicative polymerases, is capable of replicating past DNA lesions, i.e. of performing translesion synthesis (TLS). The catalytic subunit of hPolζ, hRev3, has been shown to play a critical role in DNA damage-induced mutagenesis in human cells, but less is known about the role of hRev7, the accessory subunit of hPolζ, in such mutagenesis. To address this question, we recently generated human fibroblasts with very significantly reduced levels of hRev7 protein and demonstrated that hRev7 is required to protect cells from ultraviolet(254 nm) (UV) radiation-induced cytotoxicity and mutagenesis (McNally et al., DNA Repair 7 (2008) 597-604). The goal of the present study was to determine whether hRev7 is similarly involved in the tolerance of DNA damage induced by benzo[a]pyrene diol epoxide (BPDE), the reactive form of the widespread environmental carcinogen benzo[a]pyrene. METHODS: To determine whether hRev7 also plays a role in protecting human cells from the cytotoxicity and mutagenesis induced by benzo[a]pyrene diol epoxide (BPDE), cell strains with reduced hRev7 were compared to their parental strain and a vector control strain for the effect of BPDE on cell survival, induction of mutations, and the ability to progress through the cell cycle. RESULTS: The results show that cell strains with reduced hRev7 are more sensitive to the cytotoxic effect of BPDE than the control strains, and progress through S-phase at a slower rate than the control cells following BPDE treatment, indicating that hRev7, and likely hPolζ, is required for efficient bypass of BPDE-induced DNA lesions. However, neither the frequency nor kinds of mutations induced by BPDE in cells with reduced hRev7 differ significantly from those induced in the control strains, suggesting that hPolζ is not essential for inserting nucleotides opposite BPDE-induced DNA damage. CONCLUSIONS: Taken together, our results which show that hRev7 is required for TLS past BPDE-induced DNA lesions but that it is not essential for inserting nucleotides opposite such lesions suggest a role for hPolζ in the extension step of translesion synthesis.

hRev7, putative subunit of hPolzeta, plays a critical role in survival, induction of mutations, and progression through S-phase, of UV((254nm))-irradiated human fibroblasts.

Translesion synthesis (TLS) refers to mechanisms by which specialized DNA polymerases incorporate nucleotides opposite fork-blocking lesions and extend replication until standard replicative polymerases take over. The first eukaryotic TLS polymerase discovered, S. cerevisiae Polzeta, consists of catalytic subunit Rev3 and non-catalytic subunit Rev7. Human homologs of these two proteins have been identified. Studies by Lawrence, Maher, and colleagues comparing UV((254nm))-irradiated human fibroblast cell strains expressing high levels of hRev3 antisense to their normal parental strains demonstrated that there was no difference in cell survival, but that the frequency of UV-induced mutations in the derivative strains was 10-fold lower than that of the parental strains, indicating that hRev3 plays a critical role in such mutagenesis. To examine the role of hRev7 in TLS, we generated human fibroblasts expressing hRev7 siRNA, identified two derivative cell strains with significantly reduced levels of hRev7, and compared them to their parental strain and a vector control for cell survival, induction of mutations, and ability to traverse the cell cycle following exposure to UV radiation. Cells with reduced hRev7 were approximately 2-times more sensitive to UV-induced cytotoxicity than the controls, indicating that unlike hRev3, hRev7 plays a protective role for cells exposed to UV radiation. When these cell strains were assayed for the frequency of mutations induced by UV in their HPRT gene, cell stains with reduced hRev7 were 5-times less sensitive to UV-induced mutagenesis than control strains. In addition, when these four strains were synchronized at the G1/S border, released from the block, UV-irradiated, and allowed to traverse the cell cycle, the rate of progression through S-phase of the cell strains with reduced hRev7 was significantly slower than that of the control strains. These data strongly support the hypothesis that hRev7 is required for TLS past UV-photoproducts, and together with hRev3, comprise hPolzeta.