Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:5.99.1.2 (topoisomerase)
 9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Obligatory homologous recombination (HR) is required for chiasma formation and chromosome segregation in meiosis I. Meiotic HR is initiated by DNA double-strand breaks (DSBs), generated by Spo11, a homologue of the archaebacterial topoisomerase subunit Top6A. In Saccharomyces cerevisiae, Rad50, Mre11 and Com1/Sae2 are essential to process an intermediate of the cleavage reaction consisting of Spo11 covalently linked to the 5' termini of DNA. While Rad50 and Mre11 also confer genome stability to vegetative cells and are well conserved in evolution, Com1/Sae2 was believed to be fungal-specific. Here, we identify COM1/SAE2 homologues in all eukaryotic kingdoms. Arabidopsis thaliana Com1/Sae2 mutants are sterile, accumulate AtSPO11-1 during meiotic prophase and fail to form AtRAd51 foci despite the presence of unrepaired DSBs. Furthermore, DNA fragmentation in AtCom1 is suppressed by eliminating AtSPO11-1. In addition, AtCOM1 is specifically required for mitomycin C resistance. Interestingly, we identified CtIP, an essential protein interacting with the DNA repair machinery, as the mammalian homologue of Com1/Sae2, with important implications for the molecular role of CtIP.
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PMID:A novel plant gene essential for meiosis is related to the human CtIP and the yeast COM1/SAE2 gene. 1800 98

For a cancer cell to resist treatment with drugs that trap topoisomerases covalently on the DNA, the topoisomerase must be removed. In this study, we provide evidence that the Schizosaccharomyces pombe Rad32(Mre11) nuclease activity is involved in the removal of both Top2 from 5' DNA ends as well as Top1 from 3' ends in vivo. A ctp1(CtIP) deletion is defective for Top2 removal but overproficient for Top1 removal, suggesting that Ctp1(CtIP) plays distinct roles in removing topoisomerases from 5' and 3' DNA ends. Analysis of separation of function mutants suggests that MRN-dependent topoisomerase removal contributes significantly to resistance against topoisomerase-trapping drugs. This study has important implications for our understanding of the role of the MRN complex and CtIP in resistance of cells to a clinically important group of anticancer drugs.
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PMID:Distinct requirements for the Rad32(Mre11) nuclease and Ctp1(CtIP) in the removal of covalently bound topoisomerase I and II from DNA. 1915 Apr 33

Topoisomerase inhibitors such as camptothecin and etoposide are used as anti-cancer drugs and induce double-strand breaks (DSBs) in genomic DNA in cycling cells. These DSBs are often covalently bound with polypeptides at the 3' and 5' ends. Such modifications must be eliminated before DSB repair can take place, but it remains elusive which nucleases are involved in this process. Previous studies show that CtIP plays a critical role in the generation of 3' single-strand overhang at "clean" DSBs, thus initiating homologous recombination (HR)-dependent DSB repair. To analyze the function of CtIP in detail, we conditionally disrupted the CtIP gene in the chicken DT40 cell line. We found that CtIP is essential for cellular proliferation as well as for the formation of 3' single-strand overhang, similar to what is observed in DT40 cells deficient in the Mre11/Rad50/Nbs1 complex. We also generated DT40 cell line harboring CtIP with an alanine substitution at residue Ser332, which is required for interaction with BRCA1. Although the resulting CtIP(S332A/-/-) cells exhibited accumulation of RPA and Rad51 upon DNA damage, and were proficient in HR, they showed a marked hypersensitivity to camptothecin and etoposide in comparison with CtIP(+/-/-) cells. Finally, CtIP(S332A/-/-)BRCA1(-/-) and CtIP(+/-/-)BRCA1(-/-) showed similar sensitivities to these reagents. Taken together, our data indicate that, in addition to its function in HR, CtIP plays a role in cellular tolerance to topoisomerase inhibitors. We propose that the BRCA1-CtIP complex plays a role in the nuclease-mediated elimination of oligonucleotides covalently bound to polypeptides from DSBs, thereby facilitating subsequent DSB repair.
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PMID:Collaborative action of Brca1 and CtIP in elimination of covalent modifications from double-strand breaks to facilitate subsequent break repair. 2010 9

The tumor suppressor BRCA1 functions in DNA homologous recombination, and mutations in BRCA1 increase the risk of breast and ovarian cancers. RAP80 is a component of BRCA1-containing complexes that is required for recruitment of BRCA1 to sites of DNA damage. To evaluate the role of RAP80 in DNA damage repair, we genetically disrupted both RAP80 alleles in the recombinogenic avian DT40 cell line. The resulting RAP80(-/-) cells were proficient at homologous recombination and nonhomologous end-joining (NHEJ), but were specifically sensitized to the topoisomerase II inhibitor etoposide. Notably, doubly mutant RAP80(-/-)BRCA1(-/-) cells were more sensitive to etoposide than were BRCA1(-/-) cells, revealing that RAP80 performs a BRCA1-independent repair function. Moreover, jointly impairing the function of CtIP, a distinct BRCA1 effector protein, rendered RAP80(-/-) cells more sensitive to etoposide compared with singly mutant cells, again illustrating a BRCA1-independent role of RAP80. Based on our findings, we propose that RAP80 exerts a specific function in repair of the topoisomerase-cleavage complex, such as the removal of covalently bound polypeptides from double-strand break ends independently of BRCA1.
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PMID:RAP80 acts independently of BRCA1 in repair of topoisomerase II poison-induced DNA damage. 2095 89

Double-strand breaks in chromosomal DNA are repaired efficiently in eukaryotic cells through pathways that involve direct religation of broken ends, or through pathways that utilize an unbroken, homologous DNA molecule as a template for replication. Pathways of repair that require homology initiate with the resection of the 5' strand at the break site, to uncover the 3' single-stranded DNA that becomes a critical intermediate in single-strand annealing and in homologous strand exchange. Resection of the 5' strand is regulated to occur most efficiently in S and G(2) phases of the cell cycle when sister chromatids are present as recombination templates. The mechanisms governing resection in eukaryotes have been elusive for many years, but recent work has identified the major players in short-range processing of DNA ends as well as the extensive resection of breaks that has been observed in vivo. This review focuses on the Mre11/Rad50/Xrs2(Nbs1) complex and the Sae2(CtIP) protein and their roles in initiating both short-range and long-range resection, the effects of topoisomerase-DNA conjugates on resection in vivo, and the relationship between these factors and NHEJ proteins in regulating 5' strand resection in eukaryotic cells.
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PMID:Making the best of the loose ends: Mre11/Rad50 complexes and Sae2 promote DNA double-strand break resection. 2105 Aug 28

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) repairs topoisomerase I cleavage complexes (Top1cc) by hydrolyzing their 3'-phosphotyrosyl DNA bonds and repairs bleomycin-induced DNA damage by hydrolyzing 3'-phosphoglycolates. Yeast Tdp1 has also been implicated in the repair of topoisomerase II-DNA cleavage complexes (Top2cc). To determine whether vertebrate Tdp1 is involved in the repair of various DNA end-blocking lesions, we generated Tdp1 knock-out cells in chicken DT40 cells (Tdp1-/-) and Tdp1-complemented DT40 cells with human TDP1. We found that Tdp1-/- cells were not only hypersensitive to camptothecin and bleomycin but also to etoposide, methyl methanesulfonate (MMS), H(2)O(2), and ionizing radiation. We also show they were deficient in mitochondrial Tdp1 activity. In biochemical assays, recombinant human TDP1 was found to process 5'-phosphotyrosyl DNA ends when they mimic the 5'-overhangs of Top2cc. Tdp1 also processes 3'-deoxyribose phosphates generated from hydrolysis of abasic sites, which is consistent with the hypersensitivity of Tdp1-/- cells to MMS and H(2)O(2). Because recent studies established that CtIP together with BRCA1 also repairs topoisomerase-mediated DNA damage, we generated dual Tdp1-CtIP-deficient DT40 cells. Our results show that Tdp1 and CtIP act in parallel pathways for the repair of Top1cc and MMS-induced lesions but are epistatic for Top2cc. Together, our findings reveal a broad involvement of Tdp1 in DNA repair and clarify the role of human TDP1 in the repair of Top2-induced DNA damage.
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PMID:Tyrosyl-DNA phosphodiesterase 1 (TDP1) repairs DNA damage induced by topoisomerases I and II and base alkylation in vertebrate cells. 2237 14

The CtIP protein facilitates homology-directed repair (HDR) of double-strand DNA breaks (DSBs) by initiating DNA resection, a process in which DSB ends are converted into 3'-ssDNA overhangs. The BRCA1 tumor suppressor, which interacts with CtIP in a phospho-dependent manner, has also been implicated in DSB repair through the HDR pathway. It was recently reported that the BRCA1-CtIP interaction is essential for HDR in chicken DT40 cells. To examine the role of this interaction in mammalian cells, we generated cells and mice that express Ctip polypeptides (Ctip-S326A) that fail to bind BRCA1. Surprisingly, isogenic lines of Ctip-S326A mutant and wild-type cells displayed comparable levels of HDR function and chromosomal stability. Although Ctip-S326A mutant cells were modestly sensitive to topoisomerase inhibitors, mice expressing Ctip-S326A polypeptides developed normally and did not exhibit a predisposition to cancer. Thus, in mammals, the phospho-dependent BRCA1-CtIP interaction is not essential for HDR-mediated DSB repair or for tumor suppression.
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PMID:The interaction between CtIP and BRCA1 is not essential for resection-mediated DNA repair or tumor suppression. 2371 59

Most DNA double-strand breaks (DSBs)formed in a natural environment have chemical modifications at or near the ends that preclude direct religation and require removal or other processing so that rejoining can proceed. Free radical-mediated DSBs typically bear unligatable 3'-phosphate or 3'-phosphoglycolate termini and often have oxidized bases and/or abasic sites near the break. Topoisomerase-mediated DSBs are blocked by covalently bound peptide fragments of the topoisomerase. Enzymes capable of resolving damaged ends include polynucleotide kinase/phosphatase, which restores missing 5'-phosphates and removes 3'-phosphates; tyrosyl-DNA phosphodiesterases I and II (TDP1 and TDP2), which remove peptide fragments of topoisomerases I and II, respectively, and the Artemis and Metnase endonucleases, which can trim damaged overhangs of diverse structure. TDP1 as well as APE1 can remove 3'-phosphoglycolates and other 3' blocks, while CtIP appears to provide an alternative pathway for topoisomerase II fragment removal. Ku, a core DSB joining protein, can cleave abasic sites near DNA ends. The downstream processes of patching and ligation are tolerant of residual damage, and can sometimes proceed without complete damage removal. Despite these redundant pathways for resolution, damaged ends appear to be a significant barrier to rejoining, and their resolution may be a rate-limiting step in repair of some DSBs..
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PMID:Processing of damaged DNA ends for double-strand break repair in mammalian cells. 2423 37

The carboxy-terminal binding protein (CtBP)-interacting protein (CtIP) is known to function in 5' strand resection during homologous recombination, similar to the budding yeast Sae2 protein, but its role in this process is unclear. Here, we characterize recombinant human CtIP and find that it exhibits 5' flap endonuclease activity on branched DNA structures, independent of the MRN complex. Phosphorylation of CtIP at known damage-dependent sites and other sites is essential for its catalytic activity, although the S327 and T847 phosphorylation sites are dispensable. A catalytic mutant of CtIP that is deficient in endonuclease activity exhibits wild-type levels of homologous recombination at restriction enzyme-generated breaks but is deficient in processing topoisomerase adducts and radiation-induced breaks in human cells, suggesting that the nuclease activity of CtIP is specifically required for the removal of DNA adducts at sites of DNA breaks.
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PMID:Catalytic and noncatalytic roles of the CtIP endonuclease in double-strand break end resection. 2483 76

Repair of DNA double-strand breaks (DSBs) with complex ends poses a special challenge, as additional processing is required before DNA ligation. For example, protein-DNA adducts must be removed to allow repair by either nonhomologous end joining or homology-directed repair. Here, we investigated the processing of topoisomerase II (Top2)-DNA adducts induced by treatment with the chemotherapeutic agent etoposide. Through biochemical analysis in Xenopus laevis egg extracts, we establish that the MRN (Mre11, Rad50, and Nbs1) complex, CtIP, and BRCA1 are required for both the removal of Top2-DNA adducts and the subsequent resection of Top2-adducted DSB ends. Moreover, the interaction between CtIP and BRCA1, although dispensable for resection of endonuclease-generated DSB ends, is required for resection of Top2-adducted DSBs, as well as for cellular resistance to etoposide during genomic DNA replication.
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PMID:MRN, CtIP, and BRCA1 mediate repair of topoisomerase II-DNA adducts. 2688 Jan 99


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