A Protease Pathway for the Repair of Topoisomerase II-DNA Covalent Complexes

Despite rapid advances in the field of DNA repair, little is known about the repair of protein-DNA adducts. Previous studies have demonstrated that topoisomerase II (TopII)-DNA adducts (TopII-DNA covalent complexes) are rapidly degraded by the proteasome. It has been hypothesized that proteasomal degradation of TopII-DNA covalent adducts exposes TopII-concealed DNA double-strand breaks (DSBs) for repair. To test this hypothesis, the anticancer drug, VP-16 (etoposide), was employed to induce TopII-DNA covalent complexes in mammalian cells, and the involvement of proteasome in processing TopII-DNA covalent complexes into DSBs was investigated. Consistent with the hypothesis, VP-16-induced DSBs as monitored by neutral comet assay, as well as DNA damage signals (e.g. γ-H2AX) were significantly reduced in the presence of the proteasome inhibitor, MG132. Using both top2β knock-out mouse embryonic fibroblasts and Top2β small interfering RNA knockdown PC12 cells, as well as postmitotic neurons in which TopIIα was absent, we showed that VP-16-induced DNA damage signals were attenuated upon proteasome inhibition, suggesting the involvement of proteasome in the repair/processing of both TopIIα-DNA and TopIIβ-DNA adducts. By contrast, hydrogen peroxide-induced γ-H2AX was unaffected upon proteasome inhibition, suggesting a specific requirement of the proteasome pathway in the processing of TopII-DNA covalent complexes into DNA damage. Despite rapid advances in the field of DNA repair, little is known about the repair of protein-DNA adducts. Previous studies have demonstrated that topoisomerase II (TopII)-DNA adducts (TopII-DNA covalent complexes) are rapidly degraded by the proteasome. It has been hypothesized that proteasomal degradation of TopII-DNA covalent adducts exposes TopII-concealed DNA double-strand breaks (DSBs) for repair. To test this hypothesis, the anticancer drug, VP-16 (etoposide), was employed to induce TopII-DNA covalent complexes in mammalian cells, and the involvement of proteasome in processing TopII-DNA covalent complexes into DSBs was investigated. Consistent with the hypothesis, VP-16-induced DSBs as monitored by neutral comet assay, as well as DNA damage signals (e.g. γ-H2AX) were significantly reduced in the presence of the proteasome inhibitor, MG132. Using both top2β knock-out mouse embryonic fibroblasts and Top2β small interfering RNA knockdown PC12 cells, as well as postmitotic neurons in which TopIIα was absent, we showed that VP-16-induced DNA damage signals were attenuated upon proteasome inhibition, suggesting the involvement of proteasome in the repair/processing of both TopIIα-DNA and TopIIβ-DNA adducts. By contrast, hydrogen peroxide-induced γ-H2AX was unaffected upon proteasome inhibition, suggesting a specific requirement of the proteasome pathway in the processing of TopII-DNA covalent complexes into DNA damage. VP-16 (etoposide), a prototypic topoisomerase II (TopII) 3The abbreviations used are: TopII, topoisomerase II; TopI, topoisomerase I; ICRF-193, 4,4-(2,3-butanediyl)-bis(2,6-piperazinedione); etoposide (VP-16), demethylepipodophyllotoxin ethylidene-β-d-glucoside; MG132, carbobenzoxyl-leucinyl-leucinyl-leucinal; CHX, cycloheximide; DRB, 5,6-dichlorobenzimidazole riboside; MEF, mouse embryonic fibroblast; DSB, DNA double-strand break; DIV, day in vitro; ATM-S1981-P, ATM autophosphorylation (at Ser-1981); CGN, cerebellar granule neuron; PBS, phosphate-buffered saline; CN, cortical neuron. 3The abbreviations used are: TopII, topoisomerase II; TopI, topoisomerase I; ICRF-193, 4,4-(2,3-butanediyl)-bis(2,6-piperazinedione); etoposide (VP-16), demethylepipodophyllotoxin ethylidene-β-d-glucoside; MG132, carbobenzoxyl-leucinyl-leucinyl-leucinal; CHX, cycloheximide; DRB, 5,6-dichlorobenzimidazole riboside; MEF, mouse embryonic fibroblast; DSB, DNA double-strand break; DIV, day in vitro; ATM-S1981-P, ATM autophosphorylation (at Ser-1981); CGN, cerebellar granule neuron; PBS, phosphate-buffered saline; CN, cortical neuron. poison which stabilizes both TopIIα- and TopIIβ-DNA covalent complexes (1Willmore E. Frank A.J. Padget K. Tilby M.J. Austin C.A. Mol. Pharmacol. 1998; 54: 78-85Crossref PubMed Scopus (128) Google Scholar), is widely used as a DNA damaging agent that induces DNA double-strand breaks (DSBs) (2Baldwin E.L. Osheroff N. Curr. Med. Chem. Anticancer Agents. 2005; 5: 363-372Crossref PubMed Scopus (386) Google Scholar). However, little is known about the mechanism by which TopII-DNA covalent complexes are transformed into DSBs. In vitro, the VP-16-stabilized TopII-DNA covalent complex presumably reflects the key covalent reaction intermediate, the reversible TopII cleavage complex in which each TopII subunit is covalently linked to the 5′-phosphoryl ends of the four-base staggered DSBs (3Chen G.L. Yang L. Rowe T.C. Halligan B.D. Tewey K.M. Liu L.F. J. Biol. Chem. 1984; 259: 13560-13566Abstract Full Text PDF PubMed Google Scholar). In vivo, VP-16, as well as other TopII poisons, is known to induce DNA damage signals indicative of chromosomal DNA damage. For example, TopII poisons are known to cause G2 cell cycle arrest (4Clifford B. Beljin M. Stark G.R. Taylor W.R. Cancer Res. 2003; 63: 4074-4081PubMed Google Scholar), elevation of sister chromatid exchanges (5Pommier Y. Kerrigan D. Covey J.M. Kao-Shan C.S. Whang-Peng J. Cancer Res. 1988; 48: 512-516PubMed Google Scholar, 6Noviello E. Aluigi M.G. Cimoli G. Rovini E. Mazzoni A. Parodi S. De Sessa F. Russo P. Mutat. Res. 1994; 311: 21-29Crossref PubMed Scopus (30) Google Scholar) and chromosomal aberrations (7Degrassi F. Fiore M. Palitti F. Curr. Med. Chem. Anticancer Agents. 2004; 4: 317-325Crossref PubMed Scopus (30) Google Scholar), ATM autophosphorylation (at Ser-1981) (8Kurose A. Tanaka T. Huang X. Halicka H.D. Traganos F. Dai W. Darzynkiewicz Z. Cytometry A. 2005; 68: 1-9Crossref PubMed Scopus (69) Google Scholar), H2AX phosphorylation (at Ser-139) (8Kurose A. Tanaka T. Huang X. Halicka H.D. Traganos F. Dai W. Darzynkiewicz Z. Cytometry A. 2005; 68: 1-9Crossref PubMed Scopus (69) Google Scholar), NFκB activation (9Boland M.P. Fitzgerald K.A. O'Neill L.A. J. Biol. Chem. 2000; 275: 25231-25238Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 10Andriollo M. Favier A. Guiraud P. Arch. Biochem. Biophys. 2003; 413: 75-82Crossref PubMed Scopus (16) Google Scholar), and p53 stabilization (11Karpinich N.O. Tafani M. Rothman R.J. Russo M.A. Farber J.L. J. Biol. Chem. 2002; 277: 16547-16552Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). However, how TopII-concealed DSBs are converted into DNA damage that is recognizable by the DNA damage repair system is not clear. A previous study has shown that TopII poisons induce proteasome-mediated degradation of TopII, a process referred to as TopII down-regulation (12Mao Y. Desai S.D. Ting C.Y. Hwang J. Liu L.F. J. Biol. Chem. 2001; 276: 40652-40658Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). TopII down-regulation was shown to be transcription-dependent and exhibits a preference for the TopIIβ isozyme. This phenomenon of proteasome-dependent degradation of TopII is highly reminiscent of proteasome-dependent degradation of topoisomerase I (TopI) induced by the TopI poison, camptothecin (13Desai S.D. Li T.K. Rodriguez-Bauman A. Rubin E.H. Liu L.F. Cancer Res. 2001; 61: 5926-5932PubMed Google Scholar, 14Desai S.D. Zhang H. Rodriguez-Bauman A. Yang J.M. Wu X. Gounder M.K. Rubin E.H. Liu L.F. Mol. Cell. Biol. 2003; 23: 2341-2350Crossref PubMed Scopus (121) Google Scholar). It has been hypothesized that, similar to TopI cleavage complexes, TopII cleavage complexes are degraded by the proteasome to reveal topoisomerase-concealed DNA strand breaks for repair (12Mao Y. Desai S.D. Ting C.Y. Hwang J. Liu L.F. J. Biol. Chem. 2001; 276: 40652-40658Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). In this study, we have tested the hypothesis that proteasome degradation of TopII cleavage complexes transforms TopII-concealed strand breaks into DSBs that are recognizable by the DNA damage signaling pathways. Materials—4,4-(2,3-Butanediyl)-bis(2,6-piperazinedione) (ICRF-193) was purchased from ICN Biomedicals. Etoposide (VP-16), carbobenzoxyl-leucinyl-leucinyl-leucinal (MG132), cycloheximide (CHX), 5,6-dichlorobenzimidazole 1-β-d-ribofuranoside (DRB), aphidicolin, and 4′,6-diamidino-2-phenyindole were purchased from Sigma. Lactacystin was purchased from A.G. Scientific, Inc. The pancaspase inhibitor, benzyloxycarbonyl-VAD-fluoromethyl ketone (Z-VAD-FMK), was purchased from Promega Corp. Neurobasal medium and B27 supplement were purchased from Invitrogen Corp. The anti-ATM-S1981-P antibody was purchased from Rockland Immunochemicals. Anti-γ-H2AX antibodies were purchased from Trevigen and Upstate Biotechnology. The anti-ATM-S1981-P monoclonal antibody was purchased from Cell Signaling Technology, Inc. The anti-TopIIα and TopIIβ antibodies were purchased from Santa Cruz Biotechonology, Inc. Isolation of top2β+/- and top2β-/- Mouse Embryonic Fibroblasts (MEFs)—Embryonic day-12.5 (E12.5) mouse embryos (top2β+/- and top2β-/- (15Lyu Y.L. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7123-7128Crossref PubMed Scopus (94) Google Scholar)) were dissected free of brains and livers, finely minced, and then suspended in trypsin-EDTA. The cell suspensions were incubated at 37 °C for 5 min, followed by termination with Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. The single cell suspensions were centrifuged to obtain cell pellets which were then re-suspended in fresh culture medium and plated in culture dishes. MEFs were then transformed with SV40 large T-antigen by co-transfection a SV40 T-antigen plasmid (pAN2) and a neor plasmid (at a ratio of 4:1). 48 h post-transfection, cells were selected with G418 (500 μg/ml) for 10 to 15 days. Transformed cells were then cloned. Comet Assay—Wild type transformed MEFs were pretreated with 2 μm MG132 for 30 min, followed by co-treatment with 250 μm VP-16 for 1.5 h. Cells were then washed three times with fresh medium (with or without 2 μm MG132 as indicated) and incubated for additional 30 min (for reversal of topoisomerase II cleavage complexes). For comet assay, treated cells were scraped from plates and pelletted, followed by re-suspension in 1× PBS (10,000 cells/ml). 50 μl of the cell suspension was then mixed with 500 μl 0.5% low melting point agarose at 37 °C. 75 μl of the cell/agarose mixture was transferred onto glass slides. Slides were then immersed in prechilled lysis buffer (2.5 m NaCl, 100 mm EDTA, 10 mm Tris, pH 10.0, 1% Triton X-100, and 10% Me2SO) for 1 h, followed by equilibration in 1× Tris borate-EDTA buffer for 30 min. Slides were electrophoresed in 1× Tris borate-EDTA at 1.5 volt/cm for 5 min and stained with Vistra Green (Amersham Biosciences). Images were visualized under a fluorescence microscope and captured with a CCD camera. The comet tail moment was determined using the Comet Assay IV software (Perceptive Instruments), and the mean ± S.E. of the comet tail moment was obtained from ∼50 cells for each treatment group. Statistical analysis of the mean comet tail moments was performed using Student's t test. TopIIβ Knockdown by Small Interfering RNA in PC12 Cells— Based on the 643-bp partial rat TopIIβ cDNA sequence reported in the data base (GenBank™ accession number D14046), which corresponds to the N-terminal domain of the protein, the sequence 5′-GCCCCCGTTATATCTTCAC-3′ was selected for shRNA-mediated knockdown of rat TopIIβ. The duplex DNA (5′-TGCCCCCGTTATATCTTCACTTCAAGAGAGTGAAGATATAACGGGGGCTTTTTC-3′) was made and cloned into the LentiLox 3.7 vector (obtained from Dr. Van Parijs, Massachusetts Institute of Technology, Cambridge, MA). The Top2β shRNA LentiLox 3.7 DNA was then mixed with the ViralPower™ Packaging Mix (containing pLP1, pLP2, and pLP/VSVG DNAs, which provide necessary proteins for virus production) and used for transfection into 293T cells with Lipofectamine™ 2000 (Invitrogen) to generate the lentiviral stock. Cultured PC12 cells were infected with rat Top2β shRNA lentivirus. Single colonies were isolated and characterized. The PC12-I4 clone was shown to express reduced levels of Top2β mRNA and TopIIβ protein. The PC12-C4 clone was isolated from control lentivirus (vector only)-infected PC12 cells. Neuronal Cultures—Cerebellar granule neurons (CGNs) were isolated as described by Levi et al. (16Levi G. Aloisi F. Ciotti M.T. Gallo V. Brain Res. 1984; 290: 77-86Crossref PubMed Scopus (389) Google Scholar) from postnatal day-8 (P8) Sprague-Dawley rats. Briefly, cerebella were isolated free of meninges and blood vessels in basal medium Eagle buffered with 20 mm pH at °C. were then and incubated in medium for 15 min at 37 °C. I the were then to The cell suspension was using a followed by at for 10 min. The cell pellets were in medium supplemented with B27 supplement mm mm and and 100 followed by a culture and glass were with neurons were isolated from mouse embryos the as described or were plated onto glass in a and cells for 2 day in were treated with VP-16 or camptothecin for 1 h followed by with in PBS at °C for 10 min. neurons were with PBS and then incubated with PBS 10% fetal calf Triton and for 30 min at For antibody neurons were incubated with anti-ATM-S1981-P to 500 or to antibody in PBS 1% fetal calf Triton and at °C. with PBS, neurons were incubated with antibody on glass were then washed with PBS followed by with The glass were with medium were captured using a CCD on a fluorescence was performed as described N. H. Li T.K. Liu L.F. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). Briefly, in buffer were for 10 min and then by proteins were transferred onto were immersed in for 1 h, followed by with specific antibody were then washed with mm pH mm NaCl, and incubated with the antibody for 1 h. antibody was by or the (for using VP-16 of TopII and DNA studies have demonstrated that TopII poisons induce proteasome-mediated degradation of TopII in mammalian cells (12Mao Y. Desai S.D. Ting C.Y. Hwang J. Liu L.F. J. Biol. Chem. 2001; 276: 40652-40658Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). as shown in VP-16 induced a degradation of TopIIβ to a in transformed Consistent with obtained from previous degradation of TopIIβ was by co-treatment with the proteasome MG132 suggesting the involvement of proteasome in TopIIβ It has been that proteasomal degradation of TopII cleavage complexes a repair which TopII-DNA covalent complexes into DSBs (12Mao Y. Desai S.D. Ting C.Y. Hwang J. Liu L.F. J. Biol. Chem. 2001; 276: 40652-40658Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). To test this we the of DSBs in MEFs using neutral comet in the presence and of the proteasome MG132. shown in and VP-16 μm for 1.5 treatment induced a in comet tail moment with treatment t suggesting of DSBs by with the proteasome MG132 this VP-16 t suggesting the involvement of the proteasome in the of DSBs by In the comet the of DSBs TopII cleavage complexes, a cleavage complex in fresh medium without was to the comet the of H2AX which as a for DSBs Mol. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). shown in γ-H2AX was induced by VP-16 treatment in MEFs as by with suggesting the presence of DSBs in the of VP-16-induced γ-H2AX was significantly reduced by co-treatment with the proteasome inhibitor, MG132, with the of proteasome in processing TopII-DNA covalent complexes into DSBs. of in the of TopIIα-DNA into DNA is known to induce both TopIIα-DNA and TopIIβ-DNA covalent complexes (1Willmore E. Frank A.J. Padget K. Tilby M.J. Austin C.A. Mol. Pharmacol. 1998; 54: 78-85Crossref PubMed Scopus (128) Google Scholar). The DSBs induced by VP-16 be to the processing of or both covalent complexes in transformed To the of the TopII in the of DSBs by VP-16, γ-H2AX was in TopIIβ knockdown PC12 cells and the control PC12 cells treated with shown in VP-16 induced a similar of γ-H2AX in PC12-C4 as with that in PC12-I4 cells, suggesting that the γ-H2AX is to the presence of TopIIα-DNA covalent This was by studies in top2β knock-out shown in VP-16 induced a similar of γ-H2AX in top2β-/- MEFs as with that in top2β+/- the proteasome inhibitor, MG132, reduced the γ-H2AX by about both top2β+/- and top2β-/- MEFs treated with 100 μm suggesting that the proteasome is in the processing of TopIIα-DNA covalent complexes into DSBs. of in the of TopIIβ-DNA into DNA TopIIβ-DNA covalent complexes be by proteasome into were used for shown in in of on plates induced as by not and a rapid of TopIIα for 1 day in the TopIIα in to a In VP-16 h of was shown to induce degradation of TopIIβ VP-16-induced TopIIβ degradation in postmitotic was by co-treatment with the proteasome inhibitor, MG132 suggesting the involvement of the proteasome in TopIIβ cleavage not was shown to TopIIβ suggesting the involvement of not in TopIIβ with previous in cells (12Mao Y. Desai S.D. Ting C.Y. Hwang J. Liu L.F. J. Biol. Chem. 2001; 276: 40652-40658Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). VP-16 induced DNA damage signals as ATM autophosphorylation (at and γ-H2AX in postmitotic as by using antibodies autophosphorylation was by co-treatment with MG132 or not with suggesting that VP-16-induced DNA damage signals the proteasome and not were obtained on the VP-16-induced γ-H2AX in postmitotic shown in VP-16-induced γ-H2AX in postmitotic was by co-treatment with TopII known to the of TopII-DNA covalent and MG132. are with the that TopIIβ-DNA covalent complexes are degraded by the proteasome in a transcription-dependent In the of and γ-H2AX DNA damage was monitored by in postmitotic using shown in 5 and VP-16 induced both and γ-H2AX in The of and γ-H2AX showed of both Consistent with the of both and γ-H2AX was by and MG132. In to MG132, specific proteasome was shown to induced by the involvement of proteasome in the activation of DNA damage induces DNA damage in postmitotic Cultured rat were treated with VP-16 in the presence or of μm ICRF-193, 2 μm MG132, 10 μm using anti-ATM-S1981-P antibody and antibody was performed as described under of and γ-H2AX in postmitotic on glass were treated with VP-16 for 1 h. were then and with anti-ATM-S1981-P and mouse cortical neurons were on glass and treated with or VP-16 for 1 h. was performed using the mouse cortical neurons were treated with 20 μm VP-16 for 1 h and then by using or To that VP-16-induced DNA damage signals is to the of TopIIβ covalent complexes, isolated from top2β+/- and top2β-/- embryos were shown in and VP-16 induced γ-H2AX in isolated from top2β+/- not top2β-/- mouse using antibody demonstrated that VP-16-induced γ-H2AX was the that TopIIβ-DNA covalent complexes are degraded by the proteasome to generate DNA damage VP-16, of H2AX in The proteasome requirement for the of DNA damage signals was in treated with VP-16 or shown in both VP-16 and induced indicative of the presence of DSBs in treated However, MG132 γ-H2AX induced by VP-16, not by suggesting that TopIIβ-DNA covalent complexes not DNA were by the proteasome to induce DNA damage signals To little is known about the mechanism for the repair of covalent protein-DNA adducts. of and in of S. N. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, J. Wu T.C. M. J. PubMed Scopus Google Scholar, S. N. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, A. B. D. A. P. PubMed Scopus Google Scholar)) have that as well as a are in from the covalent protein-DNA adducts C.A. PubMed Scopus Google Scholar, M. X. J.L. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, K. F. PubMed Scopus Google Scholar, M.J. J. S. 2005; PubMed Scopus Google Scholar). Previous studies have that a mechanism be in the repair/processing of TopII-DNA covalent adducts. It has been that proteasomal degradation of TopII-DNA covalent complexes a repair mechanism that transforms TopII-concealed DSBs into DSBs (12Mao Y. Desai S.D. Ting C.Y. Hwang J. Liu L.F. J. Biol. Chem. 2001; 276: 40652-40658Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). To test this we have the of proteasome in the of DNA damage in cells. Using neutral comet assay, we have monitored the of DSBs in MEFs in the presence and of MG132. In the presence of MG132, VP-16-induced DSBs was suggesting the involvement of proteasome in the of DSBs in cells. we have monitored the of the DNA damage γ-H2AX which is widely used as a for DSBs Mol. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). the VP-16-induced γ-H2AX in MEFs is reduced in the presence of suggesting the involvement of proteasome in of DNA damage by The TopII are known to be G. S. Austin C.A. F. Biophys. PubMed Scopus Google Scholar, K. S. M. T. S. Y. J. Biol. Chem. Full Text PDF PubMed Google Scholar, M. K. Y. Res. 1994; PubMed Scopus Google Scholar, H. M. S. N. A. K. J. PubMed Scopus Google Scholar) and covalent DNA adducts be by pathways. which is in and cells, has been in in with cell A. N. M. A. S. 2001; PubMed Scopus Google Scholar). In TopIIα as the with the for N. PubMed Scopus Google Scholar). It that TopIIα in cell cycle as DNA and S. K. 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PubMed Scopus Google Scholar). have the of proteasome in the processing of TopII covalent complexes into DNA damage. The TopII for the VP-16-induced γ-H2AX in MEFs is the similar of VP-16-induced γ-H2AX was in top2β-/- and top2β+/- we that proteasome is in the processing of TopIIα-DNA covalent complexes into DSBs. However, of proteasome with MG132 to a of the VP-16-induced γ-H2AX suggesting the of a for processing TopIIα cleavage complexes into DNA damage. TopIIα-DNA covalent complexes be by mechanism to in cell cycle as DNA TopIIα-DNA covalent complexes with P. Liu L.F. Cancer Res. Google Scholar) and a mechanism for repair. studies are necessary to the of repair of TopIIα-DNA covalent have demonstrated that proteasome is in the processing of TopIIβ-DNA covalent complexes into DNA damage. Using postmitotic neurons TopIIβ is we have shown that VP-16 induces proteasome-dependent degradation of TopIIβ. to TopIIβ degradation in of and as well as the of were and shown to be The proteasome for the of the DNA damage to be specific for VP-16 hydrogen peroxide-induced γ-H2AX is of suggesting that proteasome is for processing of TopIIβ-DNA adducts be by the shown in In this VP-16 stabilizes both TopIIα- and TopIIβ-DNA covalent complexes in the of reversible TopII cleavage degradation of TopII cleavage complexes exposes TopII-concealed DSBs which are then by the DNA damage repair the is proteasome is or in processing TopII-DNA covalent However, the specific requirement of proteasome for not hydrogen DNA damage in postmitotic neurons a of proteasome in processing TopIIβ-DNA adducts. VP-16-stabilized TopII-DNA covalent complexes are reversible Liu L.F. J. Biol. Chem. Full Text PDF PubMed Google Scholar). It has been the of reversible TopII-DNA covalent complexes with DNA as DNA RNA complexes, and Previous studies have demonstrated that degradation of TopIIβ is transcription-dependent (12Mao Y. Desai S.D. Ting C.Y. Hwang J. Liu L.F. J. Biol. Chem. 2001; 276: 40652-40658Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar), with in It that proteasomal degradation of TopIIβ-DNA covalent complexes be by with the complexes the (12Mao Y. Desai S.D. Ting C.Y. Hwang J. Liu L.F. J. Biol. Chem. 2001; 276: 40652-40658Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). However, the for proteasomal degradation of TopIIα-DNA covalent complexes studies are necessary to the mechanism for the repair of TopII DNA adducts. are to Dr. J. Wang for the top2β+/- mouse