The Ku Heterodimer and the Metabolism of Single-Ended DNA Double-Strand Breaks

1. Download : Download full-size image

     

Dual Roles for DNA Polymerase Theta in Alternative End-Joining Repair of Double-Strand Breaks in Drosophila

DNA double-strand breaks are repaired by multiple mechanisms that are roughly grouped into the categories of homology-directed repair and non-homologous end joining. End-joining repair can be further classified as either classical non-homologous end joining, which requires DNA ligase 4, or “alternative” end joining, which does not. Alternative end joining has been associated with genomic deletions and translocations, but its molecular mechanism(s) are largely uncharacterized. Here, we report that Drosophila melanogaster DNA polymerase theta (pol theta), encoded by the mus308 gene and previously implicated in DNA interstrand crosslink repair, plays a crucial role in DNA ligase 4-independent alternative end joining. In the absence of pol theta, end joining is impaired and residual repair often creates large deletions flanking the break site. Analysis of break repair junctions from flies with mus308 separation-of-function alleles suggests that pol theta promotes the use of long microhomologies during alternative end joining and increases the likelihood of complex insertion events. Our results establish pol theta as a key protein in alternative end joining in Drosophila and suggest a potential mechanistic link between alternative end joining and interstrand crosslink repair.     

Evidence for Ku70/Ku80 association with full-length RAG1

Antigen receptor genes are assembled by a site-specific DNA rearrangement process called V(D)J recombination. This process proceeds through two distinct phases: a cleavage phase in which the RAG1 and RAG2 proteins introduce DNA double-strand breaks at antigen receptor gene segments, and a joining phase in which the resulting DNA breaks are processed and repaired via the non-homologous end-joining (NHEJ) repair pathway. Genetic and biochemical evidence suggest that the RAG proteins play an active role in guiding the repair of DNA breaks introduced during V(D)J recombination to the NHEJ pathway. However, evidence for specific association between the RAG proteins and any of the factors involved in NHEJ remains elusive. Here we present evidence that two components of the NHEJ pathway, Ku70 and Ku80, interact with full-length RAG1, providing a biochemical link between the two phases of V(D)J recombination.     

Human Ku70/80 associates physically with telomerase through interaction with hTERT

Telomere length maintenance, an activity essential for chromosome stability and genome integrity, is regulated by telomerase- and telomere-associated factors. The DNA repair protein Ku (a heterodimer of Ku70 and Ku80 subunits) associates with mammalian telomeres and contributes to telomere maintenance. Here, we analyzed the physical association of Ku with human telomerase both in vivo and in vitro. Antibodies specific to human Ku proteins precipitated human telomerase in extracts from tumor cells, as well as from telomerase-immortalized normal cells, regardless of the presence of DNA-dependent protein kinase catalytic subunit. The same Ku antibodies also precipitated in vitro reconstituted telomerase, suggesting that this association does not require telomeric DNA. Moreover, Ku associated with the in vitro translated catalytic subunit of telomerase (hTERT) in the absence of telomerase RNA (hTR) or telomeric DNA. The results presented here are the first to report that Ku associates with hTERT, and this interaction may function to regulate the access of telomerase to telomeric DNA ends.     

Smoking Cessation Reverses DNA Double-Strand Breaks in Human Mononuclear Cells

Objective

Cigarette smoking is a major risk factor for atherosclerotic cardiovascular disease, which is responsible for a significant proportion of smoking-related deaths. However, the precise mechanism whereby smoking induces this pathology has not been fully delineated. Based on observation of DNA double-strand breaks (DSBs), the most harmful type of DNA damage, in atherosclerotic lesions, we hypothesized that there is a direct association between smoking and DSBs. The goal of this study was to investigate whether smoking induces DSBs and smoking cessation reverses DSBs in vivo through examination of peripheral mononuclear cells (MNCs).

Approach and Results

Immunoreactivity of oxidative modification of DNA and DSBs were increased in human atherosclerotic lesions but not in the adjacent normal area. DSBs in human MNCs isolated from the blood of volunteers can be detected as cytologically visible “foci” using an antibody against the phosphorylated form of the histone H2AX (γ-H2AX). Young healthy active smokers (n = 15) showed increased γ-H2AX foci number when compared with non-smokers (n = 12) (foci number/cell: median, 0.37/cell; interquartile range [IQR], 0.31–0.58 vs. 4.36/cell; IQR, 3.09–7.39, p<0.0001). Smoking cessation for 1 month reduced the γ-H2AX foci number (median, 4.44/cell; IQR, 4.36–5.24 to 0.28/cell; IQR, 0.12–0.53, p<0.05). A positive correlation was noted between γ-H2AX foci number and exhaled carbon monoxide levels (r = 0.75, p<0.01).

Conclusions

Smoking induces DSBs in human MNCs in vivo, and importantly, smoking cessation for 1 month resulted in a decrease in DSBs to a level comparable to that seen in non-smokers. These data reinforce the notion that the cigarette smoking induces DSBs and highlight the importance of smoking cessation.     

Double-strand break repair by Ku70 requires heterodimerization with Ku80 and DNA binding functions.

Heterodimers of the 70 and 80 kDa Ku autoantigens (Ku70 and Ku80) activate the DNA-dependent protein kinase (DNA-PK). Mutations in any of the three subunits of this protein kinase (Ku70, Ku80 and DNA-PKcs) lead to sensitivity to ionizing radiation (IR) and to DNA double-strand breaks, and V(D)J recombination product formation defects. Here we show that the IR repair, DNA end binding and DNA-PK defects in Ku70-/- embryonic stem cells can be counteracted by introducing epitope-tagged wild-type Ku70 cDNA. Truncations and chimeras of Ku70 were used to identify the regions necessary for DNA end binding and IR repair. Site-specific mutational analysis revealed a core region of Ku70 responsible for DNA end binding and heterodimerization. The propensity for Ku70 to associate with Ku80 and to bind DNA correlates with the ability to activate DNA-PK, although two mutants showed that the roles of Ku70 in DNA-PK activation and IR repair are separate. Mutation of DNA-PK autophosphorylation sites and other structural motifs in Ku70 showed that these sites are not necessary for IR repair in vivo. These studies reveal Ku70 features required for double-strand break repair.     

Bin1 Interacts with and Restrains the DNA End-Binding Protein Complex Ku

The Bin1 gene encodes a BAR adapter protein that suppresses cancer by poorly defined mechanisms. In an effort to gain insights, we identified cellular proteins that formed biochemical complexes with Bin1 protein. Here we report that Bin1 physically binds to Ku, a DNA end-binding protein that functions in telomere maintenance, apoptosis, and DNA repair after genotoxic stress. Both Ku70 and Ku80 were purified from human and murine cell extracts using the Bin1 BAR domain as an affinity matrix. A BAR domain mutation which destroys antioncogenic activity completely abolished Ku binding, supporting functional relevance. To further evaluate its meaning, we investigated interactions between the Bin1 homolog hob1+ and the Ku homologs pku70+ and pku80+ in fission yeast. Notably, deleting pku70+ or pku80+ relieved the survival defect displayed by hob1&Delta; cells after treatment with the DNA damaging agent phleomycin, suggesting that hob1+ may restrain Ku. Consistent with this notion, telomere length was altered in hob1&Delta; cells. The potential relevance of Bin1-Ku interaction to cancer are discussed in light of these findings.     

Sources of DNA Double-Strand Breaks and Models of Recombinational DNA Repair

DNA is subject to many endogenous and exogenous insults that impair DNA replication and proper chromosome segregation. DNA double-strand breaks (DSBs) are one of the most toxic of these lesions and must be repaired to preserve chromosomal integrity. Eukaryotes are equipped with several different, but related, repair mechanisms involving homologous recombination, including single-strand annealing, gene conversion, and break-induced replication. In this review, we highlight the chief sources of DSBs and crucial requirements for each of these repair processes, as well as the methods to identify and study intermediate steps in DSB repair by homologous recombination.     

Three-dimensional structure of the human DNA-PKcs/Ku70/Ku80 complex assembled on DNA and its implications for DNA DSB repair

DNA-PKcs is a large (approximately 470 kDa) kinase that plays an essential role in the repair of DNA double-strand breaks (DSBs) by nonhomologous end joining (NHEJ). DNA-PKcs is recruited to DSBs by the Ku70/Ku80 heterodimer, with which it forms the core of a multiprotein complex that promotes synapsis of the broken DNA ends. We have purified the human DNA-PKcs/Ku70/Ku80 holoenzyme assembled on a DNA molecule. Its three-dimensional (3D) structure at approximately 25 Angstroms resolution was determined by single-particle electron microscopy. Binding of Ku and DNA elicits conformational changes in the FAT and FATC domains of DNA-PKcs. Dimeric particles are observed in which two DNA-PKcs/Ku70/Ku80 holoenzymes interact through the N-terminal HEAT repeats. The proximity of the dimer contacts to the likely positions of the DNA ends suggests that these represent synaptic complexes that maintain broken DNA ends in proximity and provide a platform for access of the various enzymes required for end processing and ligation.     

A novel small molecule inhibitor of the DNA repair protein Ku70/80

Non-Homologous End-Joining (NHEJ) is the predominant pathway for the repair of DNA double strand breaks (DSBs) in human cells. The NHEJ pathway is frequently upregulated in several solid cancers as a compensatory mechanism for a separate DSB repair defect or for innate genomic instability, making this pathway a powerful target for synthetic lethality approaches. In addition, NHEJ reduces the efficacy of cancer treatment modalities which rely on the introduction of DSBs, like radiation therapy or genotoxic chemotherapy. Consequently, inhibition of the NHEJ pathway can modulate a radiation- or chemo-refractory disease presentation. The Ku70/80 heterodimer protein plays a pivotal role in the NHEJ process. It possesses a ring-shaped structure with high affinity for DSBs and serves as the first responder and central scaffold around which the rest of the repair complex is assembled. Because of this central position, the Ku70/80 dimer is a logical target for the disruption of the entire NHEJ pathway. Surprisingly, specific inhibitors of the Ku70/80 heterodimer are currently not available. We here describe an in silico, pocket-based drug discovery methodology utilizing the crystal structure of the Ku70/80 heterodimer. We identified a novel putative small molecule binding pocket and selected several potential inhibitors by computational screening. Subsequent biological screening resulted in the first identification of a compound with confirmed Ku-inhibitory activity in the low micro-molar range, capable of disrupting the binding of Ku70/80 to DNA substrates and impairing Ku-dependent activation of another NHEJ factor, the DNA-PK_CS kinase. Importantly, this compound synergistically sensitized human cell lines to radiation treatment, indicating a clear potential to diminish DSB repair. The chemical scaffold we here describe can be utilized as a lead-generating platform for the design and development of a novel class of anti-cancer agents.     

Human RECQ1 and RECQ4 Helicases Play Distinct Roles in DNA Replication Initiation

Cellular and biochemical studies support a role for all five human RecQ helicases in DNA replication; however, their specific functions during this process are unclear. Here we investigate the in vivo association of the five human RecQ helicases with three well-characterized human replication origins. We show that only RECQ1 (also called RECQL or RECQL1) and RECQ4 (also called RECQL4) associate with replication origins in a cell cycle-regulated fashion in unperturbed cells. RECQ4 is recruited to origins at late G₁, after ORC and MCM complex assembly, while RECQ1 and additional RECQ4 are loaded at origins at the onset of S phase, when licensed origins begin firing. Both proteins are lost from origins after DNA replication initiation, indicating either disassembly or tracking with the newly formed replisome. Nascent-origin DNA synthesis and the frequency of origin firing are reduced after RECQ1 depletion and, to a greater extent, after RECQ4 depletion. Depletion of RECQ1, though not that of RECQ4, also suppresses replication fork rates in otherwise unperturbed cells. These results indicate that RECQ1 and RECQ4 are integral components of the human replication complex and play distinct roles in DNA replication initiation and replication fork progression in vivo.The RecQ helicases are a family of DNA-unwinding enzymes essential for the maintenance of genome integrity in all kingdoms of life. Five RecQ enzymes have been found in human cells: RECQ1 (also called RECQL or RECQL1), BLM (RECQ2 or RECQL3), WRN (RECQ3 or RECQL2), RECQ4 (RECQL4), and RECQ5 (RECQL5) (3, 7). Here we refer to these helicases as RECQ1, RECQ4, and RECQ5, without the “L” that is present in the official gene names. Mutations in the BLM, WRN, and RECQ4 genes are linked to Bloom syndrome (BS), Werner syndrome (WS), and the subset of Rothmund-Thomson syndrome (RTS) patients at high risk of developing osteosarcomas, respectively (19, 31, 71). RECQ4 mutations have also been associated with RAPADILINO and Baller-Gerold syndrome (56, 61). Although these disorders are all associated with inherent genomic instability and cancer predisposition, they show distinct clinical features, suggesting that BLM, WRN, and RECQ4 are involved in different aspects of DNA metabolism. However, the molecular events underlying the pathogenesis of BS, WS, and RTS remain obscure. Mutations in the remaining two human RecQ helicase genes, RECQ1 and RECQ5, have not as yet been identified as causes of either genomic instability or heritable cancer predisposition disorders.Several lines of evidence suggest that RecQ helicases play an important role in DNA replication control (3, 10). In particular, RecQ helicases are thought to facilitate replication by preserving the integrity of stalled replication forks and by remodeling or repairing damaged or collapsed forks to allow the resumption of replication. Consistent with these ideas, several investigators have shown that primary fibroblasts from BS, WS, and RTS patients and RecQ5-deficient mouse embryonic fibroblasts all show differential hypersensitivity to agents that perturb DNA replication (12, 14, 26, 29). Moreover, BLM and WRN are recruited to DNA replication forks after replicative stress, and DNA fiber track analyses have shown that both BLM and WRN are required for normal fork progression after DNA damage or replication arrest (11-13, 47, 54). In particular, BLM in conjunction with DNA topoisomerase III and two other accessory proteins, RMI-1 and RMI-2, has been shown to catalyze the resolution of double-Holliday-junction recombination intermediates to generate noncrossover products. This dissolution reaction could play an important role in the error-free recombinational repair of damaged or stalled forks during S phase (57, 67). WRN also appears to promote error-free repair by contributing to the resolution of gene conversion events to generate noncrossover products (46). In line with the above observations, WRN and BLM can be found associated with replication foci or other DNA damage response proteins in damaged cells. In contrast, in unperturbed cells, a majority of each protein is found in the nucleolus (WRN) or associated with PML bodies (BLM) (5, 37, 62).RECQ4 has also been implicated in DNA replication. Recent studies have shown that hypomorphic mutants of the Drosophila melanogaster homolog of human RECQ4, DmRECQ4, have reduced DNA replication-dependent chorion gene amplification (65). These findings are thus consistent with a postulated role for Xenopus laevis RECQ4 (XRECQ4) in the initiation of DNA replication (39, 48). The N terminus of XRECQ4 bears homology to the N termini of the yeast proteins Sld2 (Saccharomyces cerevisiae [budding yeast]) and DRC1 (Schizosaccharomyces pombe [fission yeast]), which play a central role, in association with budding yeast Dpb11 and the fission yeast homolog Cut5/Rad4, in the establishment of DNA replication forks (38, 41, 63). Consistently, the N terminus of XRECQ4 has been shown to interact with the X. laevis variant of Cut5, and XRECQ4 depletion severely perturbs DNA replication initiation in X. laevis egg extracts (39, 48). The notion that the function of XRECQ4 is evolutionarily conserved in mammals is supported by the observations that the human protein can complement its Xenopus counterpart in cell-free assays for replication initiation and that depletion of human RECQ4 inhibits cellular proliferation and DNA synthesis (39, 48). Moreover, deletion of the N-terminal region of mouse RECQ4 has been shown to be an embryonic lethal mutation (27). These observations suggest that vertebrate RECQ4 might be a functional homolog of Sld2/DRC11, although its precise function during replication initiation and progression is not known. Recent results, published while this work was in progress, indicate that human RECQ4 interacts with the MCM replicative complex during replication initiation and that this interaction is regulated by CDK phosphorylation of RECQ4 (69). These findings, together with our results below, provide clues to the mechanism regulating RECQ4 interaction with the replication machinery.RECQ1 is the most abundant of the human RecQ helicases and was the first of the human RecQ proteins to be discovered on the basis of its potent ATPase activity (50). Despite this, little is known about the cellular functions of RECQ1, and no human disease associations have been identified to date. Recent studies have shown that RECQ1 is involved in the maintenance of genome integrity and that RECQ1 depletion affects cellular proliferation (51). Moreover, biochemical studies have shown that RECQ1 and BLM display distinct substrate specificities, indicating that these helicases are likely to perform nonoverlapping functions (43). These results suggest an important—though as yet mechanistically ill-defined—role for RECQ1 in cell cycle progression and/or DNA repair (52).In order to better delineate the role of human RecQ helicases in DNA replication, we investigated the in vivo interactions of all five human RecQ enzymes with three well-characterized human DNA replication origins in quantitative chromatin immunoprecipitation (ChIP) assays. We also determined how nascent-origin-dependent DNA synthesis, chromatin binding of replication proteins, origin firing frequency, and replication fork rates were altered by depleting specific human RecQ helicase proteins. We found that only two of the five human RecQ helicases, RECQ1 and RECQ4, specifically interact with origins in unperturbed cells. Our results provide new mechanistic insight into the distinct roles of human RECQ1 and RECQ4 in DNA replication initiation and in replication fork progression.     

Integrated analysis reveals FOXA1 and Ku70/Ku80 as targets of ivermectin in prostate cancer

Ivermectin is a widely used antiparasitic drug and shows promising anticancer activity in various cancer types. Although multiple signaling pathways modulated by ivermectin have been identified in tumor cells, few studies have focused on the exact target of ivermectin. Herein, we report the pharmacological effects and targets of ivermectin in prostate cancer. Ivermectin caused G0/G1 cell cycle arrest, induced cell apoptosis and DNA damage, and decreased androgen receptor (AR) signaling in prostate cancer cells. Further in vivo analysis showed ivermectin could suppress 22RV1 xenograft progression. Using integrated omics profiling, including RNA-seq and thermal proteome profiling, the forkhead box protein A1 (FOXA1) and non-homologous end joining (NHEJ) repair executer Ku70/Ku80 were strongly suggested as direct targets of ivermectin in prostate cancer. The interaction of ivermectin and FOXA1 reduced the chromatin accessibility of AR signaling and the G0/G1 cell cycle regulator E2F1, leading to cell proliferation inhibition. The interaction of ivermectin and Ku70/Ku80 impaired the NHEJ repair ability. Cooperating with the downregulation of homologous recombination repair ability after AR signaling inhibition, ivermectin increased intracellular DNA double-strand breaks and finally triggered cell death. Our findings demonstrate the anticancer effect of ivermectin in prostate cancer, indicating that its use may be a new therapeutic approach for prostate cancer.Subject terms: Target identification, Endocrine cancer     

Expression and chromosome location of hamster Ku70 and Ku80

Ku proteins play an important role in DNA double-strand break (DSB) repair, chromosome maintenance, and growth regulation. To understand the fundamental characteristics of Ku proteins, we examined the electrophoretic mobility and expression of hamster Ku70 and Ku80 and determined the chromosome locations of their genes. The electrophoretic mobility of hamster Ku proteins are different from that of human Ku proteins. No significant changes in the quantity of Ku proteins were observed in CHO-K1 cells treated with 10 Gy of ionizing radiation, suggesting that both proteins are expressed constitutively in amounts adequate to repair DNA DSBs. The chromosome locations of the Ku genes were determined by direct R-banding fluorescence in situ hybridization. The Ku70 gene was localized to Syrian hamster chromosome 4qa4.1--> qa4.2 and Chinese hamster chromosome 2p3.1, and the Ku80 gene was localized to Syrian hamster chromosome 4qb5--> qb6.1 and Chinese hamster chromosome 2p3.5-->p3.6. These results provide clues to the biological functions of Ku, as well as useful information for constructing comparative chromosome maps between hamsters and other mammalian species, including human, mouse, and rat.     

ATR-Chk1 Axis Protects BCR/ABL Leukemia Cells from the Lethal Effect of DNA Double-Strand Breaks

BCR/ABL-positive leukemia cells accumulated more replication-dependent DNA double-strand breaks (DSBs) than normal counterparts after treatment with cisplatin and MMC, as assessed by pulse field gel electrophoresis (PFGE) and neutral comet assay. In addition, leukemia cells could repair these lesions more efficiently than normal cells and eventually survive genotoxic treatment. Elevated levels of drug-induced DSBs in leukemia cells were associated with higher activity of ATR kinase, and enhanced phosphorylation of histone H2AX on serine 139 (&gamma;-H2AX). &gamma;-H2AX eventually started to disappear in BCR/ABL cells, while continued to increase in parental cells. In addition, the expression and ATR-mediated phosphorylation of Chk1 kinase on serine 345 were often more abundant in BCR/ABL-positive leukemia cells than normal counterparts after genotoxic treatment. Inhibition of ATR kinase by caffeine but not Chk1 kinase by indolocarbazole inhibitor, SB218078 sensitized BCR/ABL leukemia cells to MMC in a short-term survival assay. Nevertheless, both caffeine and SB218078 enhanced the genotoxic effect of MMC in a long-term clonogenic assay. This effect was associated with the abrogation of transient accumulation of leukemia cells in S and G2/M cell cycle phases after drug treatment. In conclusion, ATR - Chk1 axis was strongly activated in BCR/ABL-positive cells and contributed to the resistance to DNA cross-linking agents causing numerous replication-dependent DSBs.