DNA topoisomerases are  enzymes  capable  of altering  the  topological  conformation  of  DNA  by inducing  transient  single  (Topoisomerase  I)  and double   strand   (Topoisomerase   II)   breaks

Bohnet, S.

Lavin, M. F.

Schroeder, A. L.

English

Kansas State University

Fungal Genetics Reports Volume 36 Article 19 Topoisomerase activity assays in Neurospora A. L. Schroeder M. F. Lavin S. Bohnet Follow this and additional works at: https://newprairiepress.org/fgr This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. Recommended Citation Schroeder, A. L., M.F. Lavin, and S. Bohnet (1989) "Topoisomerase activity assays in Neurospora," Fungal Genetics Reports: Vol. 36, Article 19. https://doi.org/10.4148/1941-4765.1516 This Regular Paper is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Fungal Genetics Reports by an authorized administrator of New Prairie Press. For more information, please contact cads@k-state.edu. Topoisomerase activity assays in Neurospora Abstract DNA topoisomerases are enzymes capable of altering the topological conformation of DNA by inducing transient single (Topoisomerase I) and double strand (Topoisomerase II) breaks. This regular paper is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol36/iss1/19 Table 1. SEGREGATION PATTERNS THAT  CAN BE ASSIGNED A CHROMOSOME END Note: Ml4 has been determined to be allelic with 012 even though 012 cannot be resolvedfrom Ml2 on these gels, because 012 derives from the previously characterized VR telomere(Schechtman,  1987, op. cit.)Note added in proof: C. Myers and R. Metzenberg  (pers. comm.; Fungal Genet. Newsl.36:pp-pp)  have determined that the unassigned segregation pattern listed in the table as"09, M9 " derives from the telomere at VIIL. Supported by NSF grant DCB-8415000. ---Department of Biology, Syracuse University, Syracuse, NY 13244. (Present address: USDA,APHIS, 6505 Belcrest Road, Hyattsville, MD 20782).Schroeder, A.L.^1, M.F. Lavin^2 andS. Bohnet^1Topoisomerase activity assaysin NeurosporaDNA topoisomerases  are enzymes capable ofaltering the topological conformation of DNA byinducing transient single (Topoisomerase I) anddouble strand (Topoisomerase II) breaks. TheseThese activities appear to be involved in allaspects of DNA metabolism including replication,recombination, transcription and repair. Alleukaryotes  appear to have very similar topoiso-merase activities (Wang, J.C. 1985 Ann. Rev. Biochem. 54:665-698). We have found thatboth topoisomerase activities are easily detected in Neurospora and appear to be concen-trated in the nucleus.Changes in topoisomerase II actitivy has been proposed to be a basic defect in thehuman genetic disease, Ataxia  telangiectasia (A.T.). Activities both lower and higherthan in normal cells have been observed (Davies, S.M. et al. 1989 Nut. Acid. Res.17:1337-1352; Mohamed, R. et al. 1988 Biochem. Biophys. Res. Comm. 149:233-238) but theseresults are confounded by the effects of tissue type and transformation on topoisomeraseII levels, as well as the large variability in activity in normal controls. Two mutagen-sensitive mutants of Neurospora, uvs-6 and mus-9 with the classical cellular propertiesof A.T.: X-ray and bleomycin sensitivity, chromosomal instability and failure of X-irra-diation or bleomycin to cause normal inhibition of DNA synthesis, provide a model forunderstanding the cause of these changes without the problems of mammalian systems (Koga,S.J. and A.L. Schroeder 1987 Mutat. Res. 183:139-148). Therefore, topoisomerase activitywas examined in these mutants. Wild type and uvs-2, a mutagen-sensitive mutant withnormal chromosomal stability and DNA synthesis repression, served as controls.METHODS: General--The wild type, 74-OR23-lA,  and mutant strains have been described(Perkins, D.D. et al. 1982 Microbiol.  Rev. 46:426-570). All mutant strains have beenbackcrossed several times to standard Oak Ridge wild-type strains. General methods andmedia for growing and harvesting conidia were those of SchroederMol. Gen. Genet.(Schroeder, A.L. 1970107:291-304). For the germinating conidia used for all extracts,minimal medium containing  10^7 conidia/ml  was incubated for four hours at 3O°C with 250rpm shaking.  These conditions give 80 to 95% germination in wild type. Extracts--Forwhole cell extracts, germinating conidia were washed and resuspended at a concentrationof 5 x 10^9/ml in 0.5 ml extraction buffer containing 50 mM Tris-Cl pH 7.5, 25 mM KCl, 3mM MgCl2,  0.25M  sucrose, 1 mM  PMSF, 2 mM DTT and 1 mM  EDTA (Mohamed  et al.  1987). Theywere transferred to a Mini-Bead-Beater (Biospec Products) vial. Glass beads (0.5 mm)were added to fill the vial and enough buffer to exclude all air. After cooling on ice,the conidia were broken by four 15 sec  shakes with 45 sec  on ice between each. The glassbeads were removed using tubes with four perforations made with a dissecting needle.These were hung inside a second tube and centrifuged at 1000 x g for 5 min. The beads areretained in the upper tube. The supernatant was collected being careful not to includeany of the pelleted unbroken conidia, adjusted to 0.4 M KCL,  and centrifuged at 30,000rpm in a 50 Ti Beckman rotor for 30 min. The supernatant was brought to 70% (NH4)2SO4with a saturated  solution of (NH4)2SO4 and left at 4°C for 30 min. The precipitatedproteins were collected by centrifugation at 10,000 x g at 4°C for 15 min. andredissolved in 0.5 ml 40  mM Tris-Cl, pH 7.5, 2  mM DTT, 1  mM EDTA, 0.25 M sucrose(Dialysis Buffer). They were dialyzed against three changes of the  same buffer. Fornuclear extracts, 5  x 10^7  conidia in 0.5 ml were extracted using zirconium beads asdescribed earlier (Schroeder, A.L. 1988 Use of Neurospora to study DNA repair, in: E.C.Friedberg and P.C. Hanawalt (Eds.), DNA Repair: A laboratory manual  of research proce-dures, vol. 3, Marcel Dekker, Inc,. New York and Basel). The final nuclear pellets wereresuspended in 0.5 ml of 0.4 M KCl to extract non-histone proteins and left on ice for 1h. The extracts were centrifuged for 1 min. in a microfuge  and the supernatants  weredialyzed against dialysis buffer. Protein concentrations were determined by the methodof Lowry et al. (Lowry O.H.  et al. 1951 J. Biol. Chem. 193:265-275). Topoisomeraseassays--The assays were based on those described by Liu and Davis (Liu, L.F. and J.L.Davis 1981 Nuc. Acid. Res. 9:3979-3989).  Each 20 ul reaction contained 0-2 ug ofextracted protein, 50 mM Tris-Cl, pH 7.7, 100 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 0.5 mMwas usedEDTA and 20 ug/ml DNA substrate.  In topoisomerase I assays, covalently closed pUC19 DNAas substrate. In topoisomerase II assays, ATP was added to 1 mM and knotted P4DNA was the substrate.  Incubation was for 1 h at 37°C and the reaction was stopped bythe addition of 4 ul of 2.5% SDS; 25 mM EDTA, 30% sucrose, 0.25% bromphenol blue.  Thereaction products were analyzed by electrophoresis on 0.75% agarose gels followed bystaining with ethidium  bromide.RESULTS AND DISCUSSION: Both toposiomerase  I and II activities were easily detectedwith standard assays in both whole cell and nuclear extractsNuclear extractsof Neurospora (Fig. 1).contained at least as much activity as whole cell extracts suggestingthat these enzymes are concentrated in the nucleus in Neurospora as in other eukaryotes.Topoisomerase I activity was detected by the appearance of bands moving more slowly thanthe covalently closed circular pUC molecules (Fig. 1a). The activity was the same inwild type and mutant strains, with activity becoming detectable at 0.5 ug protein/assayin nuclear extracts. The presence of ATP,little difference in thewhich should activate topoisomerase II madeamount of activity detected. Topoisomerase II activity, whichwas assayed by the decrease in the fast moving smear of knotted P4 DNA and an increase inthe slow moving circular form, was detectable at 1.0 ug protein/assay in wild type (Fig.lb). Activity in all three mutant strains was somewhat greater than in wild type, beingpresent at 0.5 ug protein/assay in uvs-2 and uvs-6 and at 0.25 ug protein/assay in mus-9(Fig. 1b, c).  It appears that large changes in topoisomerase II activity are notnecessary for the increase in X-ray and bleomycin  sensitivity, chromosomal  instability,and failure to repress DNA synthesis after X-irradiation seen in uvs-6 and mus-9, nor dosmall increases in activity, as seen in uvs-2  necessarily lead to these properties.Wild Type- relaxed c i rc le-- supercoiledcircleuvs-6           mus-9I1 2 3 4 5 1 2 3 4 5a.                  b.Wild Type uvs-2I I1 2 3 4 5 1  2 3 4 5circle-circleFig. 1.  DNA topoisomerase activities in nuclear extracts of Neurospora. a. DNA topoiso-merase I activity in wild type measured as the unwinding of pUC19 covalently closedsupercoiled DNA.  Amounts of protein/assay: lane 1, 0 ug; lane 2, 0.01 ug; lane 3, 0.05ug; lane 4, 0.1 ug; lane 5, 0.5 ug; lane 6, 1.0 ug; lane 7, 2.0 ug; lane 8, 2.0 ug and 1mM ATP.  b. DNA topoisomerase II activity in wild type and uvs-2 measured as the unknot-ting of knotted P4 DNA in the presence of 1 mM ATP.  Amounts of protein/assay:  lane 1, 0ug; lane 2, 0.25 ug; lane 3, 0.5 ug; lane 4, 1.0 ug; lane 5 2 ug.  c. DNA topoisomeraseII activity in uvs-6 and mus-9 measured as in b.This work was supported by NSF Grant DMB-8502176 to ALS - - - ^1 Program in Genetics andCell Biology, Washington State University, Pullman, WA  99164 and ^2 Department ofBiochemistry, University of Queensland, St. Lucia, Queensland, 4067, Australia.

Topoisomerase activity assays in Neurospora

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Topoisomerase activity assays in Neurospora

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