FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1977481121> ?p ?o ?g. }

• W1977481121 endingPage "26714" @default.
• W1977481121 startingPage "26708" @default.
• W1977481121 abstract "DNA topoisomerases play essential roles in many DNA metabolic processes. It has been suggested that topoisomerases play an essential role in DNA repair. Topoisomerases can introduce DNA damage upon exposure to drugs that stabilize the covalent protein-DNA intermediate of the topoisomerase reaction. Lesions in DNA are also able to trap topoisomerase-DNA intermediates, suggesting that topoisomerases have the potential to either assist in DNA repair by locating sites of damage or exacerbating DNA damage by generation of additional damage at the site of a lesion. We have shown that overexpression of yeast topoisomerase I (TOP1) conferred hypersensitivity to methyl methanesulfonate and other DNA-damaging agents, whereas expression of a catalytically inactive enzyme did not. Overexpression of topoisomerase II did not change the sensitivity of cells to these DNA-damaging agents. Yeast cells lackingTOP1 were not more resistant to DNA damage than cells expressing wild type levels of the enzyme. Yeast topoisomerase I covalent complexes can be trapped efficiently on UV-damaged DNA. We suggest that TOP1 does not participate in the repair of DNA damage in yeast cells. However, the enzyme has the potential of exacerbating DNA damage by forming covalent DNA-protein complexes at sites of DNA damage. DNA topoisomerases play essential roles in many DNA metabolic processes. It has been suggested that topoisomerases play an essential role in DNA repair. Topoisomerases can introduce DNA damage upon exposure to drugs that stabilize the covalent protein-DNA intermediate of the topoisomerase reaction. Lesions in DNA are also able to trap topoisomerase-DNA intermediates, suggesting that topoisomerases have the potential to either assist in DNA repair by locating sites of damage or exacerbating DNA damage by generation of additional damage at the site of a lesion. We have shown that overexpression of yeast topoisomerase I (TOP1) conferred hypersensitivity to methyl methanesulfonate and other DNA-damaging agents, whereas expression of a catalytically inactive enzyme did not. Overexpression of topoisomerase II did not change the sensitivity of cells to these DNA-damaging agents. Yeast cells lackingTOP1 were not more resistant to DNA damage than cells expressing wild type levels of the enzyme. Yeast topoisomerase I covalent complexes can be trapped efficiently on UV-damaged DNA. We suggest that TOP1 does not participate in the repair of DNA damage in yeast cells. However, the enzyme has the potential of exacerbating DNA damage by forming covalent DNA-protein complexes at sites of DNA damage. methyl methanesulfonate yeast extract/peptone/dextrose/adenine medium DNA topoisomerases catalyze the interconversion of topological isomers of DNA (1Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Crossref PubMed Scopus (2086) Google Scholar). Topological changes catalyzed by these enzymes are required for a wide variety of cellular processes including transcription, replication, and chromosome segregation (2Nitiss J.L. Biochim. Biophys. Acta. 1998; 1400: 63-81Crossref PubMed Scopus (312) Google Scholar, 3Postow L. Peter B.J. Cozzarelli N.R. Bioessays. 1999; 21: 805-808Crossref PubMed Scopus (38) Google Scholar, 4Kimura K. Rybenkov V.V. Crisona N.J. Hirano T. Cozzarelli N.R. Cell. 1999; 98: 239-248Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). The importance of topoisomerases in DNA metabolism has frequently led to the suggestion that topoisomerases might play important or essential roles in DNA repair and DNA damage tolerance. However, there has been little direct evidence that topoisomerases play a direct role in the repair of DNA damage in eukaryotic cells (reviewed in Ref. 5Nitiss J.L. Nickoloff J.A. Hoekstra M.F. DNA Damage and Repair: DNA Repair in Higher Eukaryotes. Humana Press Inc., Totowa, NJ1998: 517-537Google Scholar). DNA topoisomerases are the targets of a large number of anti-cancer and anti-bacterial agents (6Rubin E.H. Li T.K. Duann P. Liu L.F. Cancer Treat. Res. 1996; 87: 243-260Crossref PubMed Scopus (37) Google Scholar, 7Burden D.A. Osheroff N. Biochim. Biophys. Acta. 1998; 1400: 139-154Crossref PubMed Scopus (501) Google Scholar). These agents stabilize a covalent intermediate where the enzyme is covalently bound to DNA through a phosphotyrosine linkage and, therefore, convert the enzyme into a DNA adduct with protein bound to the site of DNA strand breaks (8Corbett A.H. Osheroff N. Chem. Res. Toxicol. 1993; 6: 585-597Crossref PubMed Scopus (222) Google Scholar). Although the covalent intermediate is reversible, DNA metabolic processes such as replication can convert the intermediate into irreversible DNA damage. Extensive evidence has demonstrated that the DNA damage, rather than inhibition of enzyme activity, is responsible for cytotoxicity (9Nitiss J. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7501-7505Crossref PubMed Scopus (447) Google Scholar, 10Nitiss J.L. Liu Y.X. Harbury P. Jannatipour M. Wasserman R. Wang J.C. Cancer Res. 1992; 52: 4467-4472PubMed Google Scholar). Hence these agents have been termed topoisomerase poisons. Thus, topoisomerases clearly have the potential of inflicting cytotoxic DNA damage under appropriate circumstances. Recent experiments demonstrate that alterations in DNA structure are able to trap topoisomerases on DNA. Topoisomerase I can be trapped by strand discontinuities such as nicks or gaps (11Pourquier P. Pilon A.A. Kohlhagen G. Mazumder A. Sharma A. Pommier Y. J. Biol. Chem. 1997; 272: 26441-26447Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar) or by mismatched bases (12Yeh Y.C. Liu H.F. Ellis C.A. Lu A.L. J. Biol. Chem. 1994; 269: 15498-15504Abstract Full Text PDF PubMed Google Scholar). UV damage to DNA also efficiently traps eukaryotic Top1 on DNA (13Lanza A. Tornaletti S. Rodolfi C. Scanavini M. Pedrini A. J. Biol. Chem. 1996; 271: 6978-6986Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Other types of DNA damage such as abasic sites and ethenoadenine adducts also stabilize Top1 covalent complexes (14Pourquier P. Ueng L.M. Kohlhagen G. Mazumder A. Gupta M. Kohn K.W. Pommier Y. J. Biol. Chem. 1997; 272: 7792-7796Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 15Pourquier P. Bjornsti M.A. Pommier Y. J. Biol. Chem. 1998; 273: 27245-27249Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 16Pourquier P. Ueng L.M. Fertala J. Wang D. Park H.J. Essigmann J.M. Bjornsti M.A. Pommier Y. J. Biol. Chem. 1999; 274: 8516-8523Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Interestingly, there are two different mechanisms that can lead to topoisomerase I covalent complexes on DNA. UV damage, abasic sites, and mismatches all lead to a covalent complex that is not readily reversible. Other DNA lesions such as oxidized bases or benzo[a]pyrene adducts increase the rate of cleavage of the enzyme at or near the lesion but do not prevent re-ligation (17Pommier Y. Kohlhagen G. Pourquier P. Sayer J.M. Kroth H. Jerina D.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2040-2045Crossref PubMed Scopus (65) Google Scholar). This latter mechanism has also been observed for topoisomerase II at abasic sites (18Kingma P.S. Corbett A.H. Burcham P.C. Marnett L.J. Osheroff N. J. Biol. Chem. 1995; 270: 21441-21444Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 19Kingma P.S. Osheroff N. Biochim. Biophys. Acta. 1998; 1400: 223-232Crossref PubMed Scopus (81) Google Scholar). Other types of DNA damage such as UV damage inhibit topoisomerase II enzymatic activity but do not lead to increased topoisomerase II covalent complexes (20Corbett A.H. Zechiedrich E.L. Lloyd R.S. Osheroff N. J. Biol. Chem. 1991; 266: 19666-19671Abstract Full Text PDF PubMed Google Scholar). If DNA damage is able to trap topoisomerases on DNA in the same way as topoisomerase poisons, then topoisomerases may influence cell survival after DNA damage and may also influence the consequences of DNA lesions. Experiments described here test the hypothesis that the level of topoisomerases affect cell killing after DNA damage. We have taken advantage of the fact that yeast cells can tolerate different levels of both topoisomerase I and topoisomerase II. We have found that topoisomerase I overexpression greatly sensitizes yeast cells to DNA damage due to simple alkylating agents, UV light, or ionizing radiation, but overexpression of topoisomerase II does not affect yeast cell survival after exposure to these agents. These results indicate that topoisomerases can be important survival factors after DNA damage but that the enzymes do not participate directly in repair. The yeast strains used in this study are derivatives of CH335 (21Holm C. Goto T. Wang J.C. Botstein D. Cell. 1985; 41: 553-563Abstract Full Text PDF PubMed Scopus (572) Google Scholar). CH335leu was constructed by converting CH335 to leu2− by one-step gene disruption (22Alani E. Cao L. Kleckner N. Genetics. 1987; 116: 541-545Crossref PubMed Scopus (752) Google Scholar). A top1− derivative of the CH335leu was constructed by one-step gene disruption (23Nitiss J.L. Zhou J. Rose A. Hsiung Y. Gale K.C. Osheroff N. Biochemistry. 1998; 37: 3078-3085Crossref PubMed Scopus (67) Google Scholar). The top1− disruption removes the entire open reading frame of TOP1 and replaces it with the yeast LEU2 gene. The resulting strain is termed CH335top1−. Both strains CH335 and CH335leu were transfected with yCP50 or pGALyTOP1 (24Kim R.A. Wang J.C. Cell. 1989; 57: 975-985Abstract Full Text PDF PubMed Scopus (155) Google Scholar). The strains carrying yCP50 served as vector controls, whereas strains with pGALyTOP1, which expresses yeast TOP1 under the control of the yeastGAL1 promoter, were used for experiments where yeastTOP1 was overexpressed. Overexpression of humanTOP1 was accomplished using the vector pGALhTOP1 (25Bjornsti M.A. Benedetti P. Viglianti G.A. Wang J.C. Cancer Res. 1989; 49: 6318-6323PubMed Google Scholar), and expression of Escherichia coli topoisomerase I used the vector pGALECTOP1 (26Giaever G.N. Wang J.C. Cell. 1988; 55: 849-856Abstract Full Text PDF PubMed Scopus (198) Google Scholar). The three vectors for overexpressing type I topoisomerases in yeast were the gift of Dr. J. C. Wang. A vector carrying the Y727F mutation under the control of the yeastGAL1 promoter was the gift of Dr. R. Sternglanz. Sensitivity of yeast cells to MMS1 was performed as described previously (10Nitiss J.L. Liu Y.X. Harbury P. Jannatipour M. Wasserman R. Wang J.C. Cancer Res. 1992; 52: 4467-4472PubMed Google Scholar) with the following modifications. Cells were pre-grown in synthetic complete medium without uracil, with galactose as a carbon source (SC-ura/GAL). After overnight growth, cells were diluted to 2 × 106 cells/ml in fresh SC-ura/GAL, and then appropriate concentrations MMS were added. Cells were incubated for various times at 30 °C with shaking, then aliquots were removed, and diluted samples were plated to synthetic complete agar lacking uracil, with glucose as carbon source (SC-ura/Glu). Survival is expressed relative to the number of viable colonies at the time of MMS addition. For the comparison of the sensitivity oftop1− and TOP1+ cells, the appropriate strains were grown in yeast extract/peptone/dextrose/adenine (YPDA) medium as previously described (10Nitiss J.L. Liu Y.X. Harbury P. Jannatipour M. Wasserman R. Wang J.C. Cancer Res. 1992; 52: 4467-4472PubMed Google Scholar), exposed to MMS for 3 h, then plated to YPDA agar to determine the surviving fraction. All determinations were performed with at least three independent isolates; the results shown are the means ± S.E. Sensitivity to ionizing radiation was determined by pre-growing cells in SC-ura/GAL as described above to a titer of about 107cells/ml. Cells were washed in water and resuspended in water at a concentration of about 107 cells/ml. Cells were exposed to ionizing radiation using a 137Cs irradiator at a flux of 0.77 krad/min. Dilutions were then plated to SC-ura/Glu, incubated for 3 days at 30 °C, and then counted to determine the surviving fraction. Sensitivity to UV light was determined by pre-growing cells in SC-ura/GAL as described above to a titer of about 107cells/ml. Cells were diluted and plated to SC-ura/Glu. The plates were immediately exposed to UV light using a Stratalinker (Stratagene) and subsequently protected from visible light to prevent photoreactivation. Plates were incubated for 3 days at 30 °C, then plates with an appropriate number of colonies were counted, and surviving fractions compared with unirradiated plates were determined. Yeast cells were exposed to MMS under the same conditions as described for survival determinations. Total yeast RNA was isolated using the acid phenol method (27Carlson M. Botstein D. Cell. 1982; 28: 145-154Abstract Full Text PDF PubMed Scopus (926) Google Scholar). Total RNA (20 µg/sample) was separated by electrophoresis in 1% agarose gels containing 2.2 mformaldehyde. The RNA was transferred onto a nylon membrane by capillary transfer and UV cross-linked to the membrane. Hybridization was overnight at 60 °C in 0.25 mNaH2PO4, pH 7.4, 1 mm EDTA, and 7% SDS with a [32P]dCTP-labeled probe.32P-Labeled probes were prepared by random priming using a 2-kilobase BamHI fragment of the yeast RAD54gene. After hybridization, the membrane was washed 3 times at 65 °C for 15 min in 0.1× SSC (1× SSC = 0.15 m NaCl and 0.015 m sodium citrate), 0.1% SDS and then exposed to Kodak Bio-Max film at −80 °C. Yeast topoisomerase I was purified using the procedure described by Bjornsti and co-workers (28Knab A.M. Fertala J. Bjornsti M.A. J. Biol. Chem. 1995; 270: 6141-6148Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) using the plasmid pGALyTOP1 transformed into strain JEL1t1− (23Nitiss J.L. Zhou J. Rose A. Hsiung Y. Gale K.C. Osheroff N. Biochemistry. 1998; 37: 3078-3085Crossref PubMed Scopus (67) Google Scholar). One unit of topoisomerase I is defined as the amount of enzyme required to completely relax 400 ng of pUC18 in 30 min using relaxation assays as previously described (23Nitiss J.L. Zhou J. Rose A. Hsiung Y. Gale K.C. Osheroff N. Biochemistry. 1998; 37: 3078-3085Crossref PubMed Scopus (67) Google Scholar). The topoisomerase I preparation used in the experiments reported in this paper had a specific activity of 20 units/µg of protein. Formation of covalent complexes by yeast topoisomerase I was assessed using a modified K+/SDS assay. Negatively supercoiled pHOT1 DNA (TopoGen, Inc.) was used as a substrate for this assay. pHOT carries the strong topoisomerase I binding site identified by Westergaard and co-workers (29Bonven B.J. Gocke E. Westergaard O. Cell. 1985; 41: 541-551Abstract Full Text PDF PubMed Scopus (216) Google Scholar). The plasmid DNA was diluted toA 260 = 0.05 in 50 mm Tris-Cl, pH 8.5. The DNA was UV-irradiated in a 100-mm2 tissue culture dish on ice using a Stratalinker 2400 (Stratagene, 254 nm) to a final UV dose of 1000 J/m2. The DNA was then treated with an ATP-dependent exonuclease and purified using a large-construct kit protocol (Qiagen) to remove nicked DNA. The DNA was then digested with EcoRI and labeled using the Klenow fragment of DNA polymerase I and [α-32P]dCTP. Unincorporated nucleotides were removed using Chroma-spin (CLONTECH) columns. The specific activities of unirradiated and UV-irradiated DNA had similar specific activities, as determined by scintillation counting, ∼107 cpm/µg DNA. The cleavage reactions of 50 µl contained 250 ng of DNA, 10 mm Tris-Cl, pH 7.5, 70 mm KCl, 5 mmMgCl2, 0.1 mm EDTA, pH 8.0, 15 µg/ml acetylated bovine serum albumin, and 8 units of yTOP1 protein. Where indicated, samples also contained 50 µg/ml camptothecin. Reactions were incubated at 30 °C for 10 min then terminated using 1 ml of STOP buffer (1.25% SDS (w/v), 5 mm EDTA, 0.4 mg/ml salmon sperm DNA). Then 0.25 ml of 325 mm KCl was added, and samples were incubated at 65 °C for 10 min. The samples were placed on ice for 10 min, then centrifuged in an Eppendorf microcentrifuge at 8000 rpm for 10 min. The supernatant was completely removed, and samples were resuspended in 1 ml wash buffer (10 mmTris-Cl, pH 8.0, 100 mm KCl, 1 mm EDTA, 1 mg/ml salmon sperm DNA). The samples were heated to 65 °C for 10 min then held on ice for 10 min and centrifuged as before. The wash procedure was carried out a total of three times. The final precipitate was resuspended in 0.4 ml of H20. 0.1 ml was removed and added to scintillation fluid, and radioactivity was determined by scintillation counting. Negatively supercoiled pHOT1 DNA was linearized withEcoRI, diluted in water, and UV irradiated as described above. After UV treatment, the DNA was ethanol-precipitated. The topoisomerase I mobility shift assay conditions were as follows. Each reaction mixture (40 µl total volume) contained 250 ng ofEcoRI-linearized pHOT1 DNA, 10 mm Tris-Cl, pH 7.5, 2 mm KCl, 5 mm MgCl2, 0.1 mm EDTA, pH 8.0, 15 µg/ml acetylated bovine serum albumin, the indicated units of yTOP1 protein, and as indicated, 20 µg/ml camptothecin. The salt concentration was then adjusted to 70 mm with KCl for each reaction. The mixtures were incubated for 10 min at 30 °C. Reactions that were to be treated with proteinase K were stopped with 0.5 µl of 20% SDS; other sample reactions were stopped with 2 µl of 20% SDS. Proteinase K was added to a final concentration of 250 µg/ml in samples as indicated, and samples containing proteinase K were incubated overnight at 50 °C. All samples received 5 µl of a loading buffer without EDTA (60% sucrose and 0.67% Orange G) and were analyzed on a 1% agarose gel run for 400 V-h in Tris acetate EDTA (TAE) buffer. After electrophoresis, the gel was stained with ethidium bromide, and the band corresponding to free DNA was excised (to minimize interference with the hybridization). The DNA contained in the upper portion of the gel was transferred to a nylon membrane (Hybond-N, Amersham Pharmacia Biotech). The blot was hybridized overnight with a radiolabeled 500-base pairEcoRI-SspI pHOT1 DNA fragment. The blot was washed 3 times for 20 min in 0.1× SSC, 0.1% SDS at 65 °C and exposed to film. Gel shift DNA band intensities were quantitated using a STORM 860 system and an image quantification program (ImageQuant; Molecular Dynamics). The effect of topoisomerase I in the presence of DNA damage was first examined by overexpressing this enzyme inSaccharomyces cerevisiae. A plasmid carrying the yeast topoisomerase I gene under the control of the inducible GAL1promoter (24Kim R.A. Wang J.C. Cell. 1989; 57: 975-985Abstract Full Text PDF PubMed Scopus (155) Google Scholar) was transformed into yeast strain CH335. The control cells for these experiments were CH335 cells transformed with the centromeric vector yCP50. Actively growing cells were exposed to MMS in SC-ura/GAL. MMS concentrations were selected that reduced the viability of wild type cells (cells not overexpressing Top1p) to about 10–100% after 1–3-h exposures. After exposure to MMS, cells were diluted and plated to SC-ura/Glu. The results obtained with MMS are shown in Fig.1. At different concentrations of MMS, cell survival was significantly lower in cells overexpressing Top1p than in cells carrying yCP50. A similar experiment was performed using yeast cells overexpressing human topoisomerase I from the yeastGAL1 promoter. Yeast cells overexpressing hTOP1 were also more sensitive to the killing effects of MMS than cells that did not overexpress TOP1 (data not shown). Thus, the results obtained appear to be general for type 1B topoisomerases and are not due to peculiar properties of yeast topoisomerase I. The enhanced sensitivity to DNA-damaging agents was not confined to simple alkylating agents. Similar results were obtained with other types of DNA damage. Yeast cells overexpressing Top1p exposed to either UV light or ionizing radiation exhibited significantly reduced survival when compared with cells carrying the control plasmid yCP50 (Fig.2, panels A andB). Interestingly, a somewhat different pattern of sensitivity was seen with cells that express E. coli topoisomerase I, a type IA enzyme. Cells expressing E. coli topA from the yeastGAL1 promoter had slightly greater sensitivity to MMS than control cells (Fig. 3). The difference in sensitivity at 0.04% MMS was statistically significant, whereas the difference at 0.08% MMS was not statistically significant. Although expression of a type IA enzyme causes a slight increase in sensitivity to DNA-damaging agents, the effect is considerably smaller than seen when eukaryotic type 1B enzymes are overexpressed. We next examined whether the sensitization of cells by TOP1overexpression requires that the protein be active and able to cleave DNA. We introduced a plasmid carrying a mutant of TOP1 where the active site tyrosine (Tyr-727 (30Lynn R.M. Bjornsti M.A. Caron P.R. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3559-3563Crossref PubMed Scopus (111) Google Scholar)) was mutated to phenylalanine. The mutant TOP1 gene was also under the control of theGAL1 promoter (31Eng W.K. Pandit S.D. Sternglanz R. J. Biol. Chem. 1989; 264: 13373-13376Abstract Full Text PDF PubMed Google Scholar). Unlike the results obtained with the active TOP1 gene, expression of the Y727F mutant did not sensitize cells to MMS (Fig. 4). It is also noteworthy that the Y727F mutant had a similar effect on growth in the absence of MMS as overexpression of the active TOP1 gene (compare Fig. 1 and Fig. 4). This result shows that the observed sensitization by TOP1 overexpression is not due to a reduction in growth rate or decreased plating efficiency whenTOP1 is overexpressed. When topoisomerase II is transcribed at the high levels expressed from the GAL1promoter, cells are unable to successfully undergo cell division. To examine the effects of topoisomerase II overexpression in the presence of DNA-damaging agents, a plasmid containing the topoisomerase II gene in front of the constitutive promoter DED1 was transformed into yeast (10Nitiss J.L. Liu Y.X. Harbury P. Jannatipour M. Wasserman R. Wang J.C. Cancer Res. 1992; 52: 4467-4472PubMed Google Scholar). Cells were then treated with MMS, and survival was measured. The results, shown in Fig. 5, indicate that expression of the yeast TOP2 gene from theDED1 promoter does not lead to an increase in sensitivity to MMS. Similar results were obtained with UV and ionizing radiation (data not shown). These results suggest that the increase in sensitivity to DNA damage that is observed with topoisomerase overexpression is limited to type I topoisomerases. Since topoisomerase I has been implicated in transcription (32Brill S.J. DiNardo S. Voelkel-Meiman K. Sternglanz R. Nature. 1987; 326: 414-416Crossref PubMed Scopus (302) Google Scholar, 33Merino A. Madden K.R. Lane W.S. Champoux J.J. Reinberg D. Nature. 1993; 365: 227-232Crossref PubMed Scopus (324) Google Scholar), a plausible model for the effects of topoisomerase I on DNA damage response is an alteration in the expression levels of genes required for responding to DNA damage. A set of genes has been identified in yeast whose transcription is increased after DNA damage (34Ruby S.W. Szostak J.W. Mol. Cell. Biol. 1985; 5: 75-84Crossref PubMed Scopus (103) Google Scholar, 35Zhou Z. Elledge S.J. Cell. 1993; 75: 1119-1127Abstract Full Text PDF PubMed Scopus (294) Google Scholar). We examined the transcription of one such gene, RAD54, which is inducible by MMS. CH335 cells carrying pGALTOP1 were treated with MMS in either glucose or galactose, and at various times after MMS addition, aliquots were removed for RNA isolation. The RNA was electrophoresed, and after transfer to a nylon membrane, probed with full-length RAD54 DNA. The results are shown in Fig. 6. In glucose in the absence of MMS, RAD54 mRNA is barely detectable. After a 1- or 3-h exposure to 0.05% MMS, there is a clear induction of theRAD54 message. Similarly, RAD54 message is barely detectable in galactose grown cells in the absence of MMS. This suggests that overexpression of TOP1 from theGAL1 promoter by itself is insufficient to elicit the induction of RAD54. Upon the addition of MMS,RAD54 message is induced to an extent similar to that seen with glucose-grown cells. Similar results were also obtained with the gene for a subunit of the single-stranded DNA-binding protein RPA (data not shown). These results indicate that overexpression of TOP1 does not increase the sensitivity of cells to DNA-damaging agents by changing gene expression. If cells with elevated levels of topoisomerase I are hypersensitive to DNA-damaging agents, then it seemed plausible that cells completely lacking topoisomerase I have elevated resistance to DNA damage. In addition, Muller and co-workers suggested that topoisomerase I plays a role in repair, based on the detection of trapped topoisomerase I (36Subramanian D. Rosenstein B.S. Muller M.T. Cancer Res. 1998; 58: 976-984PubMed Google Scholar). We tested the sensitivity of isogenic TOP1+ and top1− cells to MMS. For clarity, only the results after a 3-h exposure to MMS are shown in Fig.7. It is clear that the sensitivity of TOP1+ and top1− cells to MMS under these conditions is the same. Similar sensitivities to UV and ionizing radiation were also found (data not shown). Our results indicate that wild type levels of topoisomerase I expression do not sensitize cells to DNA damage nor does topoisomerase I play a detectable role in repair for these DNA-damaging agents. These results agree with a previous determination of the sensitivity of top1− cells to DNA damage (37Boreham D.R. Trivedi A. Weinberger P. Mitchel R.E. Radiat. Res. 1990; 123: 203-212Crossref PubMed Scopus (19) Google Scholar). Previous reports discussed in the Introduction have shown that mammalian topoisomerase I can be trapped on DNA carrying various types of DNA damage. We next wanted to confirm that yeast topoisomerase I could also be trapped by DNA damage. We first carried out a simple assay to test whether there was preferential nicking of damaged DNA by DNA topoisomerase I. Purified yeast topoisomerase I was incubated with end-labeled DNA that was either unirradiated or UV-irradiated with 1000 J/m2. After incubation at 30 °C, the reaction was stopped with SDS, protein-DNA complexes were precipitated by the addition of excess KCl, and the samples were washed as described under “Experimental Procedures.” Topoisomerase I that was trapped as a covalent complex caused the bound DNA to precipitate, whereas free DNA remained in the supernatant. The results of this experiment are shown in Fig. 8. The addition of camptothecin to unirradiated DNA samples increased the level of topoisomerase I-DNA complexes that can be precipitated in the presence of potassium and SDS. Similarly, using DNA irradiated with 1000 J/m2 UV light in the absence of camptothecin also efficiently trapped topoisomerase I. In fact, the UV-damaged DNA trapped topoisomerase I as effectively as camptothecin. We also observed that the addition of camptothecin to samples containing irradiated DNA further increased trapping by topoisomerase I over the level seen with UV-irradiated DNA in the absence of camptothecin. To be able to examine cleavage under a wider range of conditions or with DNA substrates containing different types of DNA damage, we adapted the concept of changing the electrophoretic mobility of DNA upon protein binding to measure protein-DNA covalent complexes. In a standard electrophoretic mobility shift assay, protein and DNA are co-incubated, and then the reaction mixture is analyzed by gel electrophoresis. Detection of protein-DNA complexes when the interactions are noncovalent requires that the interaction remain stable under the electrophoresis conditions. Since we were interested in quantitating covalent protein-DNA interactions, it seemed likely that electrophoretic mobility shift would be able to readily detect topoisomerase I-DNA covalent complexes. To assess this, we examined the ability of topoisomerase I to reduce the mobility of linear DNA that was either unirradiated or irradiated with different UV doses. Fig.9 A shows the result of the electrophoresis. As can be readily seen, a weak shifted band can be observed in samples containing 6 units of topoisomerase I and unirradiated DNA. The addition of camptothecin greatly increased the intensity of the shifted band. If instead of camptothecin, UV-irradiated DNA was used, a significant increase in intensity of the shifted band was also observed. The intensities of the bands with unirradiated DNA, DNA irradiated with 1000 J/m2, or 2000 J/m2 UV is shown in Fig. 9 B. The intensity of the band is approximately linear with respect to added topoisomerase I over the range examined for all three DNA samples. The slope of the calculated linear regression is 2.8-fold higher for DNA irradiated with 1000 J/m2 than for unirradiated DNA and 5.3-fold higher for DNA irradiated with 2000 J/m2. Since the reactions were treated with SDS before electrophoresis, the interactions between topoisomerase I and damaged DNA that we detect must be covalent rather than noncovalent. To verify that the shifted bands represent protein-DNA complexes, we also treated one set of samples containing the highest amount of topoisomerase I with proteinase K before electrophoresis. Treatment with proteinase K resulted in complete loss of the shifted band whether complexes were trapped with camptothecin or UV damage. The results of Figs. 8 and 9 taken together demonstrate that UV-damaged DNA can efficiently trap topoisomerase I covalent complexes. Anti-cancer drugs such as camptothecin are able to trap a covalent intermediate of the topoisomerase I reaction, and trapping of this intermediate can interfere with DNA metabolism. It is well established that the cytotoxicity of camptothecin depends on its ability to stabilize topoisomerase I-cleavable complexes and that the degree of cytotoxicity correlates with the levels of covalent complexes (9Nitiss J. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7501-7505Crossref PubMed Scopus (447) Google Scholar). For camptothecin-induced topoisomerase I-DNA covalent complexes, processes such as DNA replication can convert the (reversible) protein-DNA lesion into an irreversible lesion (38Hsiang Y.H. Lihou M.G. Liu L.F. Cancer Res. 1989; 49: 5077-5082PubMed Google Scholar). DNA replication also can convert the single-strand break formed by topoisomerase I into a double-strand break (39Tsao Y.P. Russo A. Nyamuswa G. Silber R. Liu L.F. Cancer Res. 1993; 53: 5908-5914PubMed Google Scholar). Both the protein-DNA “adduct” and the generation of secondary double-strand breaks could contribute to camptothecin cytotoxicity. It is therefore plausible that other agents that increase the level of topoisomerase I-cleavable complexes are likely to be cytotoxic by the same mechanisms. In this paper, we demonstrate that in the presence of DNA damage, topoisomerase I also forms a stable covalent complex similar to the cleavable complexes formed in the presence of the anti-cancer drug camptothecin and that these covalent complexes enhance the cytotoxicity of DNA damage. Several trivial explanations for the hypersensitivity of cells expressing topoisomerase I to DNA-damaging agents can be readily excluded. Although cells overexpressing topoisomerase I grow more slowly than cells expressing normal levels of this enzyme, cells expressing a catalytically dead topoisomerase I also grow more slowly than wild type cells, but those cells are not hypersensitive to DNA-damaging agents. Since both yeast and human topoisomerase I expression leads to drug hypersensitivity, the sensitization to DNA-damaging agents does not arise from either a peculiar property of one of the enzymes or from the expression of a heterologous enzyme. Using two different assays we have established that yeast topoisomerase I can be efficiently trapped on UV-damaged DNA. First we used the potassium/SDS assay, which was applied to measure protein DNA-covalent complexes trapped by topoisomerase poisons both in vivo andin vitro (40Liu L.F. Rowe T.C. Yang L. Tewey K.M. Chen G.L. J. Biol. Chem. 1983; 258: 15365-15370Abstract Full Text PDF PubMed Google Scholar, 41Trask D.K. DiDonato J.A. Muller M.T. EMBO J. 1984; 3: 671-676Crossref PubMed Scopus (119) Google Scholar, 42Danks M.K. Schmidt C.A. Cirtain M.C. Suttle D.P. Beck W.T. Biochemistry. 1988; 27: 8861-8869Crossref PubMed Scopus (229) Google Scholar). Second we directly examined the levels of protein-DNA complexes using an electrophoretic mobility shift assay. Both assays gave quantitatively similar results when similar samples were examined. The potassium/SDS assay indicated a 3-fold increase in covalent complexes with 1000 J/m2 UV versus∼5-fold with the mobility shift assay. A potential advantage of the mobility shift assay is the ability to examine DNA with several different types of DNA damage. The shortcoming of the potassium SDS assay is the necessity of separately labeling each DNA that has a different type or amount of DNA damage. Other recent studies also indicate that DNA damage is able to stabilize topoisomerase I-DNA covalent complexes in vitro. Pedrini and co-workers (13Lanza A. Tornaletti S. Rodolfi C. Scanavini M. Pedrini A. J. Biol. Chem. 1996; 271: 6978-6986Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) first showed that purified topoisomerase I stably cleaves UV-damaged DNA at sites at or near UV damage (13Lanza A. Tornaletti S. Rodolfi C. Scanavini M. Pedrini A. J. Biol. Chem. 1996; 271: 6978-6986Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Pommier and co-workers (44Bhatia K. Pommier Y. Giri C. Fornace A.J. Imaizumi M. Breitman T.R. Cherney B.W. Smulson M.E. Carcinogenesis. 1990; 11: 123-128Crossref PubMed Scopus (42) Google Scholar) also find that topoisomerase I can form a stable covalent complex at the sites of several different specific DNA lesions. Earlier results had shown that factors such as DNA curvature could stimulate topoisomerase I cleavage (43Krogh S. Mortensen U.H. Westergaard O. Bonven B.J. Nucleic Acids Res. 1991; 19: 1235-1241Crossref PubMed Scopus (60) Google Scholar) and that topoisomerase I could act at sites of DNA breaks. Taken together, these results suggest that topoisomerase I action can be altered by many different changes in DNA structure. If the normal reaction of topoisomerase I at sites of DNA damage did not lead to further DNA damage, topoisomerase I could act as an efficient sensor of DNA lesions. Because the enzyme acting at damage results in a more complex lesion, the recognition of damage by topoisomerase I does not seem to be useful for promoting cell survival. Results described here that cells completely lacking topoisomerase I are not more sensitive to DNA damage than cells with wild type levels of the enzyme suggest that DNA damage recognition does not appear to be a normal indispensable role for this enzyme. Osheroff and co-workers (19Kingma P.S. Osheroff N. Biochim. Biophys. Acta. 1998; 1400: 223-232Crossref PubMed Scopus (81) Google Scholar) find that some types of DNA damage can also lead to trapping of topoisomerase II on DNA. In their studies, abasic sites greatly stimulated topoisomerase II cleavage of DNA. Since we failed to observe decreased survival in cells overexpressing topoisomerase II, the trapping of topoisomerase II by DNA damage either occurs infrequently in vivo, or cells possess an efficient system for preventing topoisomerase II-mediated DNA damage. In results to be presented elsewhere, we have found that topoisomerase II levels increase after DNA damage, which leads us to suggest that trapping of topoisomerase II in vivo is a relatively infrequent event. It may be relatively infrequent for two reasons. First, topoisomerase II cleavage is strongly inhibited by some types of DNA damage such as photoproducts induced by UV light. Second, the DNA repair systems that recognize abasic sites may be much more efficient at binding to abasic sites than topoisomerase II. If so, then cells lacking apurinic endonucleases may become sensitive to topoisomerase II dosage. We are currently testing this hypothesis. How then do cells deal with the dangerous activity of topoisomerase I when DNA is damaged? In mammalian cells, polyADP-ribose polymerase is rapidly activated by DNA strand breaks (44Bhatia K. Pommier Y. Giri C. Fornace A.J. Imaizumi M. Breitman T.R. Cherney B.W. Smulson M.E. Carcinogenesis. 1990; 11: 123-128Crossref PubMed Scopus (42) Google Scholar). A major target for poly(A)DP-ribose polymerase is topoisomerase I, and modification of topoisomerase I by this enzyme inhibits topoisomerase activity (45Ferro A.M. Olivera B.M. J. Biol. Chem. 1984; 259: 547-554Abstract Full Text PDF PubMed Google Scholar,46Ferro A.M. McElwain M.C. Olivera B.M. Cold Spring Harbor Symp. Quant. Biol. 1984; 49: 683-690Crossref PubMed Scopus (11) Google Scholar). Yeast cells apparently lack this enzyme, so some other pathway must function to attenuate topoisomerase I activity after DNA damage. The inactivation of topoisomerase I should occur fairly rapidly to prevent the formation of covalent complexes at sites of damage. Either covalent modification or targeted degradation could rapidly inactivate topoisomerase I after DNA damage. At present we do not know whether yeast topoisomerase I is rapidly degraded or whether other processes are able to inhibit the activity of the enzyme. Liu and co-workers (47Desai S.D. Liu L.F. Vazquez-Abad D. D'Arpa P. J. Biol. Chem. 1997; 272: 24159-24164Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar,48Mao Y. Sun M. Desai S.D. Liu L.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4046-4051Crossref PubMed Scopus (181) Google Scholar) show that human topoisomerase I is modified by the proteins of the ubiquitin family including small ubiquitin-related modifier after camptothecin treatment, suggesting that either degradation or inactivation of topoisomerase I can be part of the cell response to DNA damage. However, the activity and altered stability of topoisomerase I conjugated to small ubiquitin-related modifier remains to be demonstrated (49Kretz-Remy C. Tanguay R.M. Biochem. Cell Biol. 1999; 77: 299-309Crossref PubMed Scopus (31) Google Scholar). Also, down-regulation of topoisomerase I after ionizing radiation has also been reported (50Boothman D.A. Fukunaga N. Wang M. Cancer Res. 1994; 54: 4618-4626PubMed Google Scholar). There are likely other pathways that control topoisomerase I after DNA damage as well as pathways that can repair the DNA damage arising from topoisomerase I. Nash and co-workers (51Yang S.W. Burgin Jr., A.B. Huizenga B.N. Robertson C.A. Yao K.C. Nash H.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11534-11539Crossref PubMed Scopus (340) Google Scholar, 52Pouliot J.J. Yao K.C. Robertson C.A. Nash H.A. Science. 1999; 286: 552-555Crossref PubMed Scopus (316) Google Scholar) recently describe a yeast protein that can disjoin topoisomerase I covalent complexes. Since DNA damage and not just topoisomerase I poisons such as camptothecin are able to trap topoisomerase I on DNA, the enzyme described by Nash likely functions as one DNA repair system designed to deal with the ability of topoisomerase I to generate covalent complexes at the sites of damage. Our results connect topoisomerase I to pathways of DNA damage repair and DNA damage tolerance, but the connection we propose is not that topoisomerases participate in repair but, rather, as an impediment to accurate repair. It has also been found that overexpression of topoisomerase I is able to increase nonhomologous integration of transfected DNA in yeast (53Zhu J. Schiestl R.H. Mol. Cell. Biol. 1996; 16: 1805-1812Crossref PubMed Scopus (92) Google Scholar). The nonhomologous integration could arise from the action of topoisomerase at sites of endogenous DNA damage. This may suggest that topoisomerase I could play a significant role in genome destabilization after DNA damage. We thank Dr. Jerrylaine Walker for purifying yeast topoisomerase I and Mo Mehrpooya for help with cleavage assays. We also thank Drs. Rolf Sternglanz (SUNY, Stonybrook, NY), Connie Holm (University of California, San Diego, CA), and James C. Wang (Harvard University) for the gift of strains or plasmids." @default.
• W1977481121 created "2016-06-24" @default.
• W1977481121 creator A5022566853 @default.
• W1977481121 creator A5044685848 @default.
• W1977481121 creator A5060237756 @default.
• W1977481121 creator A5072102696 @default.
• W1977481121 date "2001-07-01" @default.
• W1977481121 modified "2023-10-10" @default.
• W1977481121 title "Overexpression of Type I Topoisomerases Sensitizes Yeast Cells to DNA Damage" @default.
• W1977481121 cites W1479854695 @default.
• W1977481121 cites W1523561547 @default.
• W1977481121 cites W1555798239 @default.
• W1977481121 cites W1569239054 @default.
• W1977481121 cites W1577753916 @default.
• W1977481121 cites W159023385 @default.
• W1977481121 cites W180121310 @default.
• W1977481121 cites W1975426967 @default.
• W1977481121 cites W1976156261 @default.
• W1977481121 cites W1979532948 @default.
• W1977481121 cites W1979591487 @default.
• W1977481121 cites W1987628065 @default.
• W1977481121 cites W1988516897 @default.
• W1977481121 cites W1994810131 @default.
• W1977481121 cites W2005110566 @default.
• W1977481121 cites W2005427404 @default.
• W1977481121 cites W2019897388 @default.
• W1977481121 cites W2027095809 @default.
• W1977481121 cites W2031077971 @default.
• W1977481121 cites W2031676888 @default.
• W1977481121 cites W2034660754 @default.
• W1977481121 cites W2035079536 @default.
• W1977481121 cites W2039953129 @default.
• W1977481121 cites W2042367294 @default.
• W1977481121 cites W2051249021 @default.
• W1977481121 cites W2052861371 @default.
• W1977481121 cites W2052995223 @default.
• W1977481121 cites W2054917141 @default.
• W1977481121 cites W2056382700 @default.
• W1977481121 cites W2057582459 @default.
• W1977481121 cites W2059535670 @default.
• W1977481121 cites W2070498380 @default.
• W1977481121 cites W2076418100 @default.
• W1977481121 cites W2076564663 @default.
• W1977481121 cites W2078288014 @default.
• W1977481121 cites W2081504964 @default.
• W1977481121 cites W2083338918 @default.
• W1977481121 cites W2085712777 @default.
• W1977481121 cites W2085901921 @default.
• W1977481121 cites W2093111507 @default.
• W1977481121 cites W2168762891 @default.
• W1977481121 cites W2169848221 @default.
• W1977481121 cites W2171885622 @default.
• W1977481121 cites W4236530034 @default.
• W1977481121 cites W4254536048 @default.
• W1977481121 doi "https://doi.org/10.1074/jbc.m102674200" @default.
• W1977481121 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11353773" @default.
• W1977481121 hasPublicationYear "2001" @default.
• W1977481121 type Work @default.
• W1977481121 sameAs 1977481121 @default.
• W1977481121 citedByCount "36" @default.
• W1977481121 countsByYear W19774811212012 @default.
• W1977481121 countsByYear W19774811212013 @default.
• W1977481121 countsByYear W19774811212015 @default.
• W1977481121 countsByYear W19774811212017 @default.
• W1977481121 countsByYear W19774811212019 @default.
• W1977481121 countsByYear W19774811212020 @default.
• W1977481121 countsByYear W19774811212021 @default.
• W1977481121 crossrefType "journal-article" @default.
• W1977481121 hasAuthorship W1977481121A5022566853 @default.
• W1977481121 hasAuthorship W1977481121A5044685848 @default.
• W1977481121 hasAuthorship W1977481121A5060237756 @default.
• W1977481121 hasAuthorship W1977481121A5072102696 @default.
• W1977481121 hasBestOaLocation W19774811211 @default.
• W1977481121 hasConcept C143425029 @default.
• W1977481121 hasConcept C147897179 @default.
• W1977481121 hasConcept C153911025 @default.
• W1977481121 hasConcept C185592680 @default.
• W1977481121 hasConcept C2779222958 @default.
• W1977481121 hasConcept C54355233 @default.
• W1977481121 hasConcept C552990157 @default.
• W1977481121 hasConcept C55493867 @default.
• W1977481121 hasConcept C86803240 @default.
• W1977481121 hasConcept C95444343 @default.
• W1977481121 hasConceptScore W1977481121C143425029 @default.
• W1977481121 hasConceptScore W1977481121C147897179 @default.
• W1977481121 hasConceptScore W1977481121C153911025 @default.
• W1977481121 hasConceptScore W1977481121C185592680 @default.
• W1977481121 hasConceptScore W1977481121C2779222958 @default.
• W1977481121 hasConceptScore W1977481121C54355233 @default.
• W1977481121 hasConceptScore W1977481121C552990157 @default.
• W1977481121 hasConceptScore W1977481121C55493867 @default.
• W1977481121 hasConceptScore W1977481121C86803240 @default.
• W1977481121 hasConceptScore W1977481121C95444343 @default.
• W1977481121 hasIssue "28" @default.
• W1977481121 hasLocation W19774811211 @default.
• W1977481121 hasOpenAccess W1977481121 @default.
• W1977481121 hasPrimaryLocation W19774811211 @default.
• W1977481121 hasRelatedWork W1954622523 @default.

• next