Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:5.99.1.3 (topoisomerase)
 9,911 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Exposure of mammalian cells to agents that induce DNA double-strand breaks typically results in both reciprocal and nonreciprocal chromosome translocations. Over the past decade, breakpoint junctions of a significant number of translocations and other genomic rearrangements, both in clinical tumors and in experimental models, have been analyzed at the DNA sequence level. Based on these data, reasonable inferences regarding the biochemical mechanisms involved in translocations can be drawn. In a few cases, breakpoints have been shown to correlate with sites of double-strand cleavage by agents to which the cells or patients have been exposed, including exogenous rare-cutting endonucleases, radiomimetic compounds, and topoisomerase inhibitors. These results confirm that translocations primarily reflect misjoining of the exchanged ends of two or more double-strand breaks. Many junctions show significant loss of DNA sequence at the breakpoints, suggesting exonucleolytic degradation of DNA ends prior to joining. The size and frequency of these deletions varies widely, both between experimental systems, and among individual events in a single system. Homologous recombination between repetitive DNA sequences does not appear to be a major pathway for translocations associated with double-strand breaks. Rather, the general features of the junction sequences, particularly the high frequency small terminal deletions, the apparent splicing of DNA ends at microhomologies, and gap-filling on aligned double-strand break ends, are consistent with the known biochemical properties of the classical nonhomologous end joining pathway involving DNA-dependent protein kinase, XRCC4 and DNA ligase IV. Nevertheless, cells with deficiencies in this pathway still exhibit translocations, with grossly similar junction sequences, suggesting an alternative but less conservative end joining pathway. Although evidence for participation of specific DNA end processing enzymes in formation of translocations is largely circumstantial, likely candidates include DNA polymerases lambda and mu, Artemis nuclease, polynucleotide kinase/phosphatase, tyrosyl-DNA phosphodiesterase, DNase III, Werner syndrome protein, and the Mre11/Rad50/NBS1 complex.
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PMID:Biochemical mechanisms of chromosomal translocations resulting from DNA double-strand breaks. 1682 25

To study the relationships between different DNA repair pathways, we established a set of clones in which one specific DNA repair gene was silenced using long-term RNA interference in HeLa cell line. We focus here on genes involved in either nucleotide excision repair (XPA and XPC) or nonhomologous end joining (NHEJ; DNA-PKcs and XRCC4). As expected, XPA(KD) (knock down) and XPC(KD) cells were highly sensitive to UVC. DNA-PKcs(KD) and XRCC4(KD) cells presented an increased sensitivity to various inducers of double-strand breaks (DSBs) and a 70% to 80% reduction of in vitro NHEJ activity. Long-term silencing of XPC gene expression led to an increased sensitivity to etoposide, a topoisomerase II inhibitor that creates DSBs through the progression of DNA replication forks. XPC(KD) cells also showed intolerance toward acute gamma-ray irradiation. We showed that XPC(KD) cells exhibited an altered spectrum of NHEJ products with decreased levels of intramolecular joined products. Moreover, in both XPC(KD) and DNA-PKcs(KD) cells, XRCC4 and ligase IV proteins were mobilized on damaged nuclear structures at lower doses of DSB inducer. In XPC-proficient cells, XPC protein was released from nuclear structures after induction of DSBs. By contrast, silencing of XPA gene expression did not have any effect on sensitivity to DSB or NHEJ. Our results suggest that XPC deficiency, certainly in combination with other genetic defects, may contribute to impair DSB repair.
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PMID:Long-term XPC silencing reduces DNA double-strand break repair. 1736 70

Human polynucleotide kinase (hPNK) is a bifunctional enzyme possessing a 5'-DNA kinase activity and a 3'-phosphatase activity. Studies based on cell extracts and purified proteins have indicated that hPNK can act on single-strand breaks and double-strand breaks (DSB) to restore the termini to the chemical form required for further action by DNA repair polymerases and ligases (i.e., 5'-phosphate and 3'-hydroxyl termini). These studies have revealed that hPNK can bind to XRCC4, and as a result, hPNK has been implicated as a participant in the nonhomologous end joining (NHEJ) pathway for DSB repair. We sought to confirm the role of hPNK in NHEJ in the cellular setting using a genetic approach. hPNK was stably down-regulated by RNA interference expression in M059K glioblastoma cells, which are NHEJ positive, and M059J cells, which are NHEJ deficient due to a lack of DNA-PK catalytic subunit (DNA-PKcs). Whereas depletion of hPNK significantly sensitized M059K cells to ionizing radiation, no additional sensitization was conferred to M059J cells, clearly implying that hPNK operates in the same DNA repair pathway as DNA-PKcs. On the other hand, depletion of hPNK did not increase the level of sister chromatid exchanges, indicating that hPNK is not involved in the homologous recombination DSB repair pathway. We also provide evidence that the action of hPNK in the repair of camptothecin-induced topoisomerase 1 "dead-end" complexes is independent of DNA-PKcs and that hPNK is not involved in the nucleotide excision repair pathway.
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PMID:Human polynucleotide kinase participates in repair of DNA double-strand breaks by nonhomologous end joining but not homologous recombination. 1763 72

KU70(-/-) and DNA-PKcs(-/-/-)chicken DT40 cells are reportedly highly sensitive to the DNA topoisomerase II inhibitor etoposide. Here we report that KU70 and DNA-PKcs unexpectedly function together during the induction of apoptosis after exposure to high levels of etoposide. In the presence of 100 microM etoposide, apoptosis was induced within 1 h in wild type DT40 cells but not in KU70(-/-) and DNA-PKcs(-/-/-) cells. In addition, the DNA-PK inhibitors NU7026 and wortmannin, as well as the caspase inhibitor Z-VAD-FMK, inhibited etoposide-induced apoptosis in wild type cells. Although Artemis(-/-) cells also showed defects in the etoposide-induced apoptosis, the other mutants defective in nonhomologous end-joining (NHEJ), LIG4(-/-), XRCC4(-), and XLF(-/-) cells were capable to induce apoptosis. When cells were treated with high doses of etoposide, the chromatin binding of DNA-PKcs was impaired by deletion of KU70 but not of Artemis, suggesting that KU70 acts upstream of DNA-PKcs and Artemis acts downstream of DNA-PKcs in the apoptotic pathway like the NHEJ pathway. These results suggest that the proteins involved in the early stage of NHEJ pathway including Artemis but not the downstream factors decide the cell fate by selecting apoptosis or DNA repair according to the degree of DNA damage.
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PMID:KU70/80, DNA-PKcs, and Artemis are essential for the rapid induction of apoptosis after massive DSB formation. 1867 14

DNA DSBs (double-strand breaks) are considered the most cytotoxic type of DNA lesion. They can be introduced by external sources such as IR (ionizing radiation), by chemotherapeutic drugs such as topoisomerase poisons and by normal biological processes such as V(D)J recombination. If left unrepaired, DSBs can cause cell death. If misrepaired, DSBs may lead to chromosomal translocations and genomic instability. One of the major pathways for the repair of IR-induced DSBs in mammalian cells is NHEJ (non-homologous end-joining). The main proteins required for NHEJ in mammalian cells are the Ku heterodimer (Ku70/80 heterodimer), DNA-PKcs [the catalytic subunit of DNA-PK (DNA-dependent protein kinase)], Artemis, XRCC4 (X-ray-complementing Chinese hamster gene 4), DNA ligase IV and XLF (XRCC4-like factor; also called Cernunnos). Additional proteins, including DNA polymerases mu and lambda, PNK (polynucleotide kinase) and WRN (Werner's Syndrome helicase), may also play a role. In the present review, we will discuss our current understanding of the mechanism of NHEJ in mammalian cells and discuss the roles of DNA-PKcs and DNA-PK-mediated phosphorylation in NHEJ.
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PMID:Repair of ionizing radiation-induced DNA double-strand breaks by non-homologous end-joining. 1913 41

DNA double-strand breaks (DSBs) can be repaired by one of two major pathways-non-homologous end-joining (NHEJ) and homologous recombination (HR)-depending on whether cells are in G1 or S/G2 phase, respectively. However, the mechanisms of DSB repair during M phase remain largely unclear. In this study, we demonstrate that transient treatment of M-phase cells with the chemotherapeutic topoisomerase inhibitor etoposide induced DSBs that were often associated with anaphase bridge formation and genome instability such as dicentric chromosomes. Although most of the DSBs were carried over into the next G1 phase, some were repaired during M phase. Both NHEJ and HR, in particular NHEJ, promoted anaphase-bridge formation, suggesting that these repair pathways can induce genome instability during M phase. On the other hand, C-terminal-binding protein interacting protein (CtIP) suppressed anaphase bridge formation, implying that CtIP function prevents genome instability during mitosis. We also observed M-phase-specific phosphorylation of XRCC4, a regulatory subunit of the ligase IV complex specialized for NHEJ. This phosphorylation required cyclin-dependent kinase (CDK) activity as well as polo-like kinase 1 (Plk1). A phosphorylation-defective XRCC4 mutant showed more efficient M-phase DSB repair accompanied with an increase in anaphase bridge formation. These results suggest that phosphorylation of XRCC4 suppresses DSB repair by modulating ligase IV function to prevent genome instability during M phase. Taken together, our results indicate that XRCC4 is required not only for the promotion of NHEJ during interphase but also for its M-phase-specific suppression of DSB repair.
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PMID:Canonical non-homologous end joining in mitosis induces genome instability and is suppressed by M-phase-specific phosphorylation of XRCC4. 2516 5

The repair of DNA double-strand breaks (DSB) is central to the maintenance of genomic integrity. In tumor cells, the ability to repair DSBs predicts response to radiation and many cytotoxic anti-cancer drugs. DSB repair pathways include homologous recombination and non-homologous end joining (NHEJ). NHEJ is a template-independent mechanism, yet many NHEJ repair products carry limited genetic changes, which suggests that NHEJ includes mechanisms to minimize error. Proteins required for mammalian NHEJ include Ku70/80, the DNA-dependent protein kinase (DNA-PKcs), XLF/Cernunnos and the XRCC4:DNA ligase IV complex. NHEJ also utilizes accessory proteins that include DNA polymerases, nucleases, and other end-processing factors. In yeast, mutations of tyrosyl-DNA phosphodiesterase (TDP1) reduced NHEJ fidelity. TDP1 plays an important role in repair of topoisomerase-mediated DNA damage and 3'-blocking DNA lesions, and mutation of the human TDP1 gene results in an inherited human neuropathy termed SCAN1. We found that human TDP1 stimulated DNA binding by XLF and physically interacted with XLF to form TDP1:XLF:DNA complexes. TDP1:XLF interactions preferentially stimulated TDP1 activity on dsDNA as compared to ssDNA. TDP1 also promoted DNA binding by Ku70/80 and stimulated DNA-PK activity. Because Ku70/80 and XLF are the first factors recruited to the DSB at the onset of NHEJ, our data suggest a role for TDP1 during the early stages of mammalian NHEJ.
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PMID:TDP1 promotes assembly of non-homologous end joining protein complexes on DNA. 2584 Nov 1

Smarcal1 is a SWI/SNF-family protein with an ATPase domain involved in DNA-annealing activities and a binding site for the RPA single-strand-DNA-binding protein. Although the role played by Smarcal1 in the maintenance of replication forks has been established, it remains unknown whether Smarcal1 contributes to genomic DNA maintenance outside of the S phase. We disrupted the SMARCAL1 gene in both the chicken DT40 and the human TK6 B cell lines. The resulting SMARCAL1(-/-) clones exhibited sensitivity to chemotherapeutic topoisomerase 2 inhibitors, just as nonhomologous end-joining (NHEJ) null-deficient cells do. SMARCAL1(-/-) cells also exhibited an increase in radiosensitivity in the G1 phase. Moreover, the loss of Smarcal1 in NHEJ null-deficient cells does not further increase their radiosensitivity. These results demonstrate that Smarcal1 is required for efficient NHEJ-mediated DSB repair. Both inactivation of the ATPase domain and deletion of the RPA-binding site cause the same phenotype as does null-mutation of Smarcal1, suggesting that Smarcal1 enhances NHEJ, presumably by interacting with RPA at unwound single-strand sequences and then facilitating annealing at DSB ends. SMARCAL1(-/-)cells showed a poor accumulation of Ku70/DNA-PKcs and XRCC4 at DNA-damage sites. We propose that Smarcal1 maintains the duplex status of DSBs to ensure proper recruitment of NHEJ factors to DSB sites.
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PMID:Smarcal1 promotes double-strand-break repair by nonhomologous end-joining. 2608 90