Cockayne Syndrome Group B Protein Stimulates Repair of Formamidopyrimidines by NEIL1 DNA Glycosylase
2009; Elsevier BV; Volume: 284; Issue: 14 Linguagem: Inglês
10.1074/jbc.m807006200
ISSN1083-351X
AutoresMeltem Müftüoğlu, Nadja C. de Souza‐Pinto, Arın Doğan, Maria D. Aamann, Tinna Stevnsner, Ivana Rybanská, Güldal Kırkalı, Miral Dizdaroğlu, Vilhelm A. Bohr,
Tópico(s)Acute Lymphoblastic Leukemia research
ResumoCockayne syndrome (CS) is a premature aging condition characterized by sensitivity to UV radiation. However, this phenotype does not explain the progressive neurodegeneration in CS patients. It could be due to the hypersensitivity of CSB-deficient cells to oxidative stress. So far most studies on the role of CSB in repair of oxidatively induced DNA lesions have focused on 7,8-dihydro-8-oxoguanine. This study examines the role of CSB in the repair of formamidopyrimidines 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4,6-diamino-5-formamidopyrimidine (FapyAde), which are substrates for endonuclease VIII-like (NEIL1) DNA glycosylase. Results presented here show that csb-/- mice have a higher level of endogenous FapyAde and FapyGua in DNA from brain and kidney than wild type mice as well as higher levels of endogenous FapyAde in genomic DNA and mtDNA from liver. In addition, CSB stimulates NEIL1 incision activity in vitro, and CSB and NEIL1 co-immunoprecipitate and co-localize in HeLa cells. When CSB and NEIL1 are depleted from HeLa cells by short hairpin RNA knockdown, repair of induced FapyGua is strongly inhibited. These results suggest that CSB plays a role in repair of formamidopyrimidines, possibly by interacting with and stimulating NEIL1, and that accumulation of such modifications may have a causal role in the pathogenesis of CS. Cockayne syndrome (CS) is a premature aging condition characterized by sensitivity to UV radiation. However, this phenotype does not explain the progressive neurodegeneration in CS patients. It could be due to the hypersensitivity of CSB-deficient cells to oxidative stress. So far most studies on the role of CSB in repair of oxidatively induced DNA lesions have focused on 7,8-dihydro-8-oxoguanine. This study examines the role of CSB in the repair of formamidopyrimidines 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4,6-diamino-5-formamidopyrimidine (FapyAde), which are substrates for endonuclease VIII-like (NEIL1) DNA glycosylase. Results presented here show that csb-/- mice have a higher level of endogenous FapyAde and FapyGua in DNA from brain and kidney than wild type mice as well as higher levels of endogenous FapyAde in genomic DNA and mtDNA from liver. In addition, CSB stimulates NEIL1 incision activity in vitro, and CSB and NEIL1 co-immunoprecipitate and co-localize in HeLa cells. When CSB and NEIL1 are depleted from HeLa cells by short hairpin RNA knockdown, repair of induced FapyGua is strongly inhibited. These results suggest that CSB plays a role in repair of formamidopyrimidines, possibly by interacting with and stimulating NEIL1, and that accumulation of such modifications may have a causal role in the pathogenesis of CS. Cockayne syndrome (CS) 6The abbreviations used are: CS, cockayne syndrome; BER, base excision repair; OGG1, 8-oxoguanine DNA glycosylase; 8-OH-Gua, 7,8-dihydro-8-oxoguanine; PARP1, poly(ADP-ribose) polymerase; Fpg, formamidopyrimidine DNA glycosylase; shRNA, short hairpin RNA; GC, gas chromatography; MS, mass spectrometry; LC, liquid chromatography; 5-OH-Ura, 5-hydroxyuracil; ssDNA, single-stranded DNA; Fapy, formamidopyrimidine; FapyGua, 2,6-diamino-4-hydroxy-5-Fapy; FapyAde, 4,6-diamino-5-Fapy; PBS, phosphate-buffered saline; wt, wild type; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; CSBfl, full-length CSB; AP, apurinic or apyrimidinic. is a segmental premature aging syndrome with progressive neurological degeneration (1Rapin I. Lindenbaum Y. Dickson D.W. Kraemer K.H. Robbins J.H. Neurology. 2000; 55: 1442-1449Crossref PubMed Scopus (185) Google Scholar). CS is caused by mutations in CS complementation groups A (CSA) or B (CSB) genes (2Tanaka K. Kawai K. Kumahara Y. Ikenaga M. Okada Y. Somatic Cell Genet. 1981; 7: 445-455Crossref PubMed Scopus (78) Google Scholar, 3Stefanini M. Fawcett H. Botta E. Nardo T. Lehmann A.R. Hum. Genet. 1996; 97: 418-423Crossref PubMed Scopus (76) Google Scholar). Approximately 80% of CS patients have mutations in the CSB gene, which encodes a 168-kDa protein belonging to the SWI/SNF2 family of chromatin remodeling proteins (4Troelstra C. Odijk H. de Wit J. Westerveld A. Thompson L.H. Bootsma D. Hoeijmakers J.H. Mol. Cell. Biol. 1990; 10: 5806-5813Crossref PubMed Scopus (109) Google Scholar). Cells from CS patients are hypersensitive to UV radiation-induced DNA damage, and the CSB protein is required for the transcription-coupled nucleotide excision repair of UV radiation-induced DNA lesions (cyclobutane pyrimidine dimers and 6-pyrimidine-4-pyrimidone products) (5van Oosterwijk M.F. Versteeg A. Filon R. van Zeeland A.A. Mullenders L.H. Mol. Cell. Biol. 1996; 16: 4436-4444Crossref PubMed Scopus (87) Google Scholar). CSB is also believed to play a role in transcription elongation and interacts with the RNA polymerase II elongation complex (6Iyer N. Reagan M.S. Wu K.J. Canagarajah B. Friedberg E.C. Biochemistry. 1996; 35: 2157-2167Crossref PubMed Scopus (167) Google Scholar). The molecular basis of the progressive neurological defects in CS patients, however, remains unknown; it has been proposed that neurological symptoms in CS may be due to defective repair and/or processing of oxidative DNA damage in CSB-deficient cells (7Kraemer K.H. Patronas N.J. Schiffmann R. Brooks B.P. Tamura D. DiGiovanna J.J. Neuroscience. 2007; 145: 1388-1396Crossref PubMed Scopus (305) Google Scholar). Oxidative DNA damage can be caused by endogenous and exogenous agents. Reactive oxygen species, including highly reactive hydroxyl radicals, are formed as byproducts of normal metabolism, mostly during the process of mitochondrial respiration. It has been estimated that up to 2% of all the O2 consumed by respiration may be released as reactive oxygen species (8Nohl H. Gille L. Staniek K. Biochem. Pharmacol. 2005; 69: 719-723Crossref PubMed Scopus (190) Google Scholar, 9Raha S. Robinson B.H. Am. J. Med. Genet. 2001; 106: 62-70Crossref PubMed Scopus (257) Google Scholar). The central nervous system relies exclusively on mitochondria to generate ATP through oxidative metabolism. As a result, neurons are susceptible to increased levels of oxidative stress, and elevated levels of reactive oxygen species have been implicated in the etiology of neurodegenerative diseases including Alzheimer, Parkinson, and Huntington diseases and amyotrophic lateral sclerosis (for a review, see Ref. 10Mattson M.P. Liu D. Neuromolecular Med. 2002; 2: 215-231Crossref PubMed Scopus (180) Google Scholar). Hydroxyl radicals attack DNA bases and the sugar-phosphate DNA backbone, generating modified bases and single-stranded DNA (ssDNA) breaks, respectively (11Evans M.D. Dizdaroglu M. Cooke M.S. Mutat. Res. 2004; 567: 1-61Crossref PubMed Scopus (1006) Google Scholar). Many oxidatively induced DNA lesions are mutagenic and/or cytotoxic and have been associated with aging, neurodegeneration, and carcinogenesis (for review, see Refs. 12de Souza-Pinto N.C. Bohr V.A. Int. Rev. Neurobiol. 2002; 53: 519-534Crossref PubMed Google Scholar and 13Loft S. Poulsen H.E. J. Mol. Med. 1996; 74: 297-312Crossref PubMed Scopus (822) Google Scholar). Most of these lesions are repaired by the base excision repair pathway (BER), during which lesion-specific DNA glycosylases hydrolyze the N-glycosidic bond between the modified base and the sugar moiety to release the modified base and generate an abasic (AP) site. Subsequent repair steps include cleavage of the resulting abasic site, incorporation of one or few nucleotides, trimming of the 5′ and 3′ ends, and ligation of the DNA backbone (14Wilson III, D.M. Sofinowski T.M. McNeill D.R. Front. Biosci. 2003; 8: 963-981Crossref PubMed Google Scholar). Several DNA glycosylases specifically recognize and repair oxidatively induced DNA lesions (15Ide H. Kotera M. Biol. Pharm. Bull. 2004; 27: 480-485Crossref PubMed Scopus (104) Google Scholar). The substrate specificities of DNA glycosylases are broad and, in some cases, overlapping, which may explain the absence of severe phenotypes in knock-out mice lacking 8-oxoguanine DNA glycosylase (OGG1) or endonuclease III homologue (NTH1). In contrast, mice lacking endonuclease VIII-like (NEIL1) DNA glycosylase have a combination of clinical manifestations resembling human metabolic syndrome (16Vartanian V. Lowell B. Minko I.G. Wood T.G. Ceci J.D. George S. Ballinger S.W. Corless C.L. McCullough A.K. Lloyd R.S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 1864-1869Crossref PubMed Scopus (185) Google Scholar). This phenotype suggests that NEIL1 is essential for repair of an endogenous DNA lesion or lesions that has significant biological consequences when incorrectly or incompletely repaired. Recent evidence suggests that CSB may play a role in repair of oxidatively induced DNA damage. Embryonic fibroblasts from csb-/- mice are hypersensitive to γ-irradiation and paraquat, a redox-cycling compound that generates oxidative stress (17de Waard H. de Witt J. Gorgels T.G. van den A.G. Andressoo J.O. Vermeij M. van S.H. Hoeijmakers J.H. van der Horst G.T. DNA Repair (Amst.). 2003; 2: 13-25Crossref PubMed Scopus (118) Google Scholar), and γ-irradiated human cells expressing mutated CSB have defects in repair of 7,8-dihydro-8-oxoguanine (8-OH-Gua) and 7,8-dihydro-8-oxoadenine (18Tuo J. Muftuoglu M. Chen C. Jaruga P. Selzer R.R. Brosh Jr., R.M. Rodriguez H. Dizdaroglu M. Bohr V.A. J. Biol. Chem. 2001; 276: 45772-45779Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 19Tuo J. Jaruga P. Rodriguez H. Dizdaroglu M. Bohr V.A. J. Biol. Chem. 2002; 277: 30832-30837Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Furthermore, CSB is found in a complex with OGG1, although no direct interaction between the two proteins has been identified (20Tuo J. Chen C. Zeng X. Christiansen M. Bohr V.A. DNA Repair (Amst.). 2002; 1: 913-927Crossref PubMed Scopus (87) Google Scholar). More importantly, hepatocytes, splenocytes, and kidney cells from ogg1-/-/csb-/- mice accumulate several-fold higher levels of modified purines than csb-/- mice (21Osterod M. Larsen E. Le P.F. Hengstler J.G. van der Horst G.T. Boiteux S. Klungland A. Epe B. Oncogene. 2002; 21: 8232-8239Crossref PubMed Scopus (122) Google Scholar) and have elevated levels of G → T transversion mutations (22Trapp C. Reite K. Klungland A. Epe B. Oncogene. 2007; 26: 4044-4048Crossref PubMed Scopus (41) Google Scholar). CSB also interacts with other BER enzymes, including AP endonuclease 1 (23Wong H.K. Muftuoglu M. Beck G. Imam S.Z. Bohr V.A. Wilson III, D.M. Nucleic Acids Res. 2007; 35: 4103-4113Crossref PubMed Scopus (97) Google Scholar) and poly(ADP-ribose) polymerase (PARP1) (24Thorslund T. von K.C. Harrigan J.A. Indig F.E. Christiansen M. Stevnsner T. Bohr V.A. Mol. Cell. Biol. 2005; 25: 7625-7636Crossref PubMed Scopus (97) Google Scholar). Several studies have investigated the role of CSB in repair of 8-OH-Gua. This lesion, however, may be a weak mutagen in vivo because it can be faithfully bypassed in the presence of proliferating cell nuclear antigen and replication protein A (25Maga G. Villani G. Crespan E. Wimmer U. Ferrari E. Bertocci B. Hubscher U. Nature. 2007; 447: 606-608Crossref PubMed Scopus (187) Google Scholar). Formamidopyrimidines (Fapys) 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4,6-diamino-5-formamidopyrimidine (FapyAde) are imidazole ring-opened products of purines which share a common intermediate with 8-OH-Gua and 7,8-dihydro-8-oxoadenine, respectively (11Evans M.D. Dizdaroglu M. Cooke M.S. Mutat. Res. 2004; 567: 1-61Crossref PubMed Scopus (1006) Google Scholar, 26Steenken S. Chem. Rev. 1989; 89: 503-520Crossref Scopus (1113) Google Scholar). FapyGua and FapyAde are the main substrates of human and mouse NEIL1 (27Jaruga P. Birincioglu M. Rosenquist T.A. Dizdaroglu M. Biochemistry. 2004; 43: 15909-15914Crossref PubMed Scopus (76) Google Scholar, 28Roy L.M. Jaruga P. Wood T.G. McCullough A.K. Dizdaroglu M. Lloyd R.S. J. Biol. Chem. 2007; 282: 15790-15798Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 29Hazra T.K. Izumi T. Boldogh I. Imhoff B. Kow Y.W. Jaruga P. Dizdaroglu M. Mitra S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3523-3528Crossref PubMed Scopus (432) Google Scholar) and are detected in genomic DNA at a higher level than 8-OH-Gua (30Hu J. de Souza-Pinto N.C. Haraguchi K. Hogue B.A. Jaruga P. Greenberg M.M. Dizdaroglu M. Bohr V.A. J. Biol. Chem. 2005; 280: 40544-40551Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar), suggesting that accumulation of these lesions may be of importance in the development of the severe phenotype observed in neil1-/- mice. This study examines the possible role of CSB in repair of FapyGua and FapyAde. In vivo studies show that endogenous Fapys accumulate in brain, liver, and kidney genomic DNA and liver mtDNA from csb-/- mice. In vitro studies show that CSB greatly enhances NEIL1 catalytic activity via a stimulation of the strand-incision step. CSB and NEIL1 also co-localize in HeLa cells and co-immunoprecipitate from HeLa nuclear extracts. Short hairpin RNAs (shRNA) double knockdown of CSB and NEIL1 greatly inhibits repair of FapyGua. These results suggest that NEIL1 and CSB cooperate in the repair of FapyAde and FapyGua. Recombinant Proteins-Recombinant N-terminal hemagglutinin antigen- and C-terminal His6-double-tagged human CSB protein was purified from HiFive insect cells as described previously (31Christiansen M. Stevnsner T. Modin C. Martensen P.M. Brosh Jr., R.M. Bohr V.A. Nucleic Acids Res. 2003; 31: 963-973Crossref PubMed Scopus (42) Google Scholar). Escherichia coli formamidopyrimidine DNA glycosylase (Fpg) was isolated and purified as described (32Reddy P. Jaruga P. O'Connor T. Rodriguez H. Dizdaroglu M. Protein Expression Purif. 2004; 34: 126-133Crossref PubMed Scopus (20) Google Scholar). Purified recombinant NEIL1 protein was kindly provided by Dr. Sankar Mitra (University of Texas Medical Branch, Galveston, TX). Cell Lines and shRNA Transfection-HeLa cells were maintained at 37 °C, 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Invitrogen). For knockdown experiments, the cells were transfected with 50 nm shRNA targeting either human CSB (OriGene, catalog no. TR313176), human NEIL1 (OriGene, catalog no. TR307276), or non-targeting control shRNA (OriGene) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Stable NEIL1 KD and CSB/NEIL1 double KD cells were selected and maintained in 2 μg/ml puromycin. Individual clones were selected and tested for knockdown levels of CSB and NEIL1 by Western blot analysis. The clones demonstrating the lower expression levels of CSB and NEIL1 were chosen for further analysis. For the treatments the clones were plated into 15-cm dishes (in triplicate) and incubated until 80–90% confluence was reached. The cells were exposed to 200 μm menadione (in PBS) for 30 min at 37 °C. At the end of the treatment the drug was removed, the cultures were washed twice with PBS, and the cells were immediately harvested for DNA isolation, or the culture medium was replaced, and the cells were incubated for 6 h at 37 °C for repair. After the repair period, the cultures were washed once with PBS, and the cells were harvested for DNA isolation. Preparation of Mouse Brain, Liver, and Kidney Mitochondrial or Nuclear Extracts-CSB knock-out mice (csb-/-), kindly provided by Dr. Jan Hoeijmakers (Erasmus University Medical Center, Rotterdam, Netherlands), were bred at the Gerontology Research Center Animal Facility under standard conditions. Wild type (wt) littermates were used as controls. Mice were sacrificed by cervical dislocation, and the brain, liver, and kidney were immediately removed and processed. Nuclear and mitochondrial extracts were prepared as described earlier (33de Souza-Pinto N.C. Hogue B.A. Bohr V.A. Free Radic. Biol. Med. 2001; 30: 916-923Crossref PubMed Scopus (95) Google Scholar). All experiments were approved by the Gerontology Research Center Animal Care and Use Committee and performed in accordance with National Institutes of Health Guidelines for the Care and Use of Laboratory Animals, National Institutes of Health Publication 85-23, National Institutes of Health, Bethesda, MD. Preparation of mtDNA and nDNA from Mouse Brain, Liver, and Kidney-DNA was isolated using a modification of the salting-out method (34Miller S.A. Dykes D.D. Polesky H.F. Nucleic Acids Res. 1988; 16: 1215Crossref PubMed Scopus (17626) Google Scholar). Mitochondrial or nuclear pellets from brain, liver, and kidney were suspended in 2 ml of buffer containing 210 mm mannitol, 70 mm sucrose, 10 mm Hepes-KOH, pH 7.4, 2 mm EGTA, 1 mm EDTA, 2 mm dithiothreitol, 0.15 mm spermine, and 0.75 mm spermidine. The homogenates were then diluted in lysis buffer containing 10 mm Tris-HCl, pH 8.2, 2 mm EDTA, 400 mm NaCl, 1% SDS, to an approximate protein concentration of 1 mg/ml. Two mg/ml of proteinase K (Sigma) were added, and the samples were incubated at 37 °C overnight. One-fourth volume of saturated NaCl was added, and proteins were precipitated by centrifugation at 16,100 × g for 15 min. The supernatant was recovered, and the DNA was precipitated with 2.5 volumes of 96% ethanol at -20 °C for 1–2 h. The precipitated DNA was collected by centrifugation, suspended in 5 ml of 10 mm Tris, 1 mm EDTA, and incubated with RNase A (0.1 mg/ml) at 37 °C for 1 h. The samples were then subjected to another proteinase K (0.5 mg/ml) digestion in lysis buffer at 55 °C for 1 h. After precipitation of proteins with saturated NaCl and centrifugations as above, DNA was precipitated from the supernatant with ethanol, collected by centrifugation, and dried in a SpeedVac. Analysis by Gas Chromatography/Mass Spectrometry (GC/MS) and Liquid Chromatography LC/MS-All DNA samples were blinded before measurement. DNA samples were dissolved in water. The DNA quality and concentration in each sample were determined by the UV spectrum recorded between 200 and 350 nm. The identification and quantification of 8-OH-Gua, FapyGua, and FapyAde in DNA was performed by GC/MS after hydrolysis of DNA samples by E. coli Fpg (32Reddy P. Jaruga P. O'Connor T. Rodriguez H. Dizdaroglu M. Protein Expression Purif. 2004; 34: 126-133Crossref PubMed Scopus (20) Google Scholar, 35Boiteux S. Gajewski E. Laval J. Dizdaroglu M. Biochemistry. 1992; 31: 106-110Crossref PubMed Scopus (571) Google Scholar). 50-μg aliquots of DNA samples were supplemented with aliquots of 8-OH-Gua[13C,15N3], [13C,15N3]FapyAde, and [13C,15N3]FapyGua as internal standards and then hydrolyzed with 2 μg of Fpg as described (32Reddy P. Jaruga P. O'Connor T. Rodriguez H. Dizdaroglu M. Protein Expression Purif. 2004; 34: 126-133Crossref PubMed Scopus (20) Google Scholar). After ethanol precipitation, DNA pellets and supernatant fractions were separated by centrifugation. Supernatant fractions were lyophilized, trimethylsilylated, and analyzed by GC/MS as described (32Reddy P. Jaruga P. O'Connor T. Rodriguez H. Dizdaroglu M. Protein Expression Purif. 2004; 34: 126-133Crossref PubMed Scopus (20) Google Scholar). For identification and quantification, selected-ion monitoring was used to monitor the characteristic ions of trimethylsilylated 8-OH-Gua, FapyAde, and FapyGua and their stable isotope-labeled analogues as internal standards (36Dizdaroglu M. FEBS Lett. 1993; 315: 1-6Crossref PubMed Scopus (137) Google Scholar). LC/MS was used to identify and quantify 8-OH-Gua as its nucleoside, 8-OH-dG, in DNA samples. 50-μg aliquots of DNA samples were supplemented with an aliquot of 8-OH-[15N5]dG as internal standard. Samples were hydrolyzed with nuclease P1, snake venom phosphodiesterase, and alkaline phosphatase for 24 h at 37 °C and then analyzed by LC/MS as described (27Jaruga P. Birincioglu M. Rosenquist T.A. Dizdaroglu M. Biochemistry. 2004; 43: 15909-15914Crossref PubMed Scopus (76) Google Scholar). For identification and quantification, selected-ion monitoring was used to monitor the characteristic ions of 8-OH-dG and its internal standard (37Dizdaroglu M. Jaruga P. Rodriguez H. Nucleic Acids Res. 2001; 29: E12Crossref PubMed Scopus (104) Google Scholar). The levels of 8-OH-Gua measured with GC/MS and LC/MS were essentially identical (supplemental Fig. S1). Statistical analyses of GC/MS and LC/MS results were performed using a Kruskal-Wallis test with S-Plus 7 statistical program. A p value equal to or lower than 0.05 was considered to be statistically significant. Oligodeoxynucleotides-The sequences of the oligodeoxynucleotides used here are shown in Table 1. FapyGua-containing oligodeoxynucleotides were a kind gift from Dr. Marc Greenberg (38Jiang Y.L. Wiederholt C.J. Patro J.N. Haraguchi K. Greenberg M.M. J. Org. Chem. 2005; 70: 141-149Crossref PubMed Scopus (32) Google Scholar). The oligodeoxynucleotides containing 5-OH-Ura or 8-OH-Gua were obtained from Midland Certified Reagent Co. (Midland, TX). All oligodeoxynucleotides were 5′-end-labeled using T4 polynucleotide kinase and [γ-32P]ATP as described before (39Souza-Pinto N.C. Croteau D.L. Hudson E.K. Hansford R.G. Bohr V.A. Nucleic Acids Res. 1999; 27: 1935-1942Crossref PubMed Scopus (110) Google Scholar). FapyGua-, 8-OH-Gua-, and 5-OH-Ura-containing oligodeoxynucleotides were annealed to the complementary strand in 10 mm Tris-HCl, pH 7.8, 1 mm EDTA, and 100 mm KCl by heating the samples at 90 °C for 5 min and slowly cooling to room temperature.TABLE 1Oligodeoxynucleotides used for incision assays Fg, FapyGua; Fa, FapyAde; OHU, 5-OH-Ura; OHG, 8-OH-Gua. The unpaired region in the bubble-containing oligo is underlined.Oligodeoxynucleotides and sequencesFapyGua5′-CCAGGTGCFgAAGTGGTAGCACGTCCCAT-3′3′-GGTCCACG C TTCACCATCGTGCAGGGTA-5′FapyAde5′-CGTTCAACGTGCACTFaACAGCACGTCCCAT-3′3′-GCAAGTTGCACGTGAT TGTCGTGCAGGGTA-5′Duplex 5-OH-Ura5′-GCTTAGCTTGGAATCGTATCATGTAOHUACTCGTGTGCCGTGTAGACCGTGCC-3′3′-CGAATCGAACCTTAGCATAGTACATGTGAGCACACGGCACATCTGGCACGG-5′Bubble 5-OH-Ura5′-GCTTAGCTTGGAATCGTATCATGTAOHUACTCGTGTGCCGTGTAGACCGTGCC-3′3′-CGAATCGAACCTTAGCATAGGCACCCGACAAACACGGCACATCTGGCACGG-5′8-OH-Gua5′-GCTTAGCTTGGAATCGTATCATGTAOHGACTCGTGTGCCGTGTAGACCGTGCC-3′3′-CGAATCGAACCTTAGCATAGTACATCTGAGCACACGGCACATCTGGCACGG-5′ Open table in a new tab DNA Glycosylase Assays for NEIL1-Incision of FapyGua, FapyAde, 5-OH-Ura, or 8-OH-Gua was performed in a reaction mixture (10 μl) containing 40 mm Hepes-KOH, pH 7.6, 1 mm EDTA, 2 mm dithiothreitol, 50 mm NaCl, 0.2 μg/μl bovine serum albumin, 5% glycerol, and 50 fmol of 32P-labeled DNA substrate, except for FapyAde, which contained 5 fmol. The reactions were initiated by adding NEIL1 and CSB as indicated in the figure legends 2–5. The reactions were incubated at 37 °C for 30 or 60 min and stopped with the addition of 5 μg of proteinase K and 1 μl of 10% SDS followed by 15 min of incubation at 37 °C. To promote complete strand cleavage at the abasic sites, 100 mm NaOH was added and incubated at 37 °C for 15 min. An equal amount of formamide loading dye (90% formamide, 10 mm EDTA, 0.01% bromphenol blue, 0.01% xylene cyanol) was added, and the samples were incubated at 95 °C for 5 min and resolved by electrophoresis on a 20% polyacrylamide, 7 m urea gel. Gels were visualized by PhosphorImager and analyzed using the ImageQuant software (GE HealthCare). The percentage of incision was calculated as the amount of radioactivity present in the product band relative to the total radioactivity. To measure NEIL1 AP-lyase activity, the experiments were processed as described above without incubation with NaOH. DNA Trapping Assay-DNA trapping assays were performed as described for the glycosylase assay, with the addition of freshly made 50 mm NaBH4 at the start of the reactions. After incubation at 37 °C for 2 h, the reactions were terminated by adding 5 μl of 5× SDS-PAGE sample buffer, and the samples were heated at 95 °C for 5 min. Trapped protein-DNA complexes were separated in 12% SDS-PAGE. The gels were visualized using PhosphorImager and analyzed using the ImageQuant software (GE Healthcare). Construction of Recombinant CSB Fragments and CSB Point Mutant Proteins-CSB-(2–341) and CSB-(514–986) fragments were purified as previously described (24Thorslund T. von K.C. Harrigan J.A. Indig F.E. Christiansen M. Stevnsner T. Bohr V.A. Mol. Cell. Biol. 2005; 25: 7625-7636Crossref PubMed Scopus (97) Google Scholar). Briefly, the fragments were cloned into the pTriEx-4 Neo vector (Novagen), which encodes N-terminal His and S tags and C-terminal herpes simplex virus and His tags. The two fragments were overexpressed in E. coli and purified using the His and S tags. Construction of pcDNA3.1-CSBE646Q and CSBQ942E plasmids, making stable transfected CS1AN cell lines and purification of CSB wild type and mutant proteins, were done as described previously (31Christiansen M. Stevnsner T. Modin C. Martensen P.M. Brosh Jr., R.M. Bohr V.A. Nucleic Acids Res. 2003; 31: 963-973Crossref PubMed Scopus (42) Google Scholar, 40Muftuoglu M. Selzer R. Tuo J. Brosh Jr., R.M. Bohr V.A. Gene (Amst.). 2002; 283: 27-40Crossref PubMed Scopus (39) Google Scholar, 41Selzer R.R. Nyaga S. Tuo J. May A. Muftuoglu M. Christiansen M. Citterio E. Brosh Jr., R.M. Bohr V.A. Nucleic Acids Res. 2002; 30: 782-793Crossref PubMed Scopus (62) Google Scholar). Co-immunoprecipitation Assay-HeLa nuclear extracts were prepared as described previously (42Muftuoglu M. Sharma S. Thorslund T. Stevnsner T. Soerensen M.M. Brosh Jr., R.M. Bohr V.A. Nucleic Acids Res. 2006; 34: 295-304Crossref PubMed Scopus (39) Google Scholar) and were precleared for 1 h with rProtein G-agarose beads (Invitrogen). Extracts (250 μg each) were incubated with either 4 μg of rabbit anti-CSB antibody (Santa Cruz Biotechnology), 4 μg of rabbit anti-NEIL1 antibody (Calbiochem), or 4 μg of rabbit IgG (Santa Cruz Biotechnology) as a negative control for 16 h at 4 °C. Each sample was then incubated with rProtein G-agarose beads (30 μl) at 4 °C for 1 h. Bound proteins were eluted by boiling in SDS sample buffer for 5 min and analyzed by Western blotting with goat anti-CSB (1:1000; Santa Cruz Biotechnology) or goat anti-NEIL1 (1:500; Santa Cruz Biotechnology) antibodies for 16 h at 4 °C followed by chemiluminescent analysis (Pierce). Immunofluorescence Staining-HeLa cells were plated on four-well Lab-Tek Chamber Slides and grown for 24 h. Cells were washed 3 times with PBS and fixed in freshly prepared 4% paraformaldehyde in PBS for 15 min at room temperature. The cells were then washed 3 times in PBS and permeabilized in cold 0.25% Triton X-100 solution in PBS for 10 min on ice followed by three washes in PBS. Cells were blocked for unspecific protein binding using 1% bovine serum albumin in PBS for 30 min at room temperature. After blocking, primary antibodies diluted in washing buffer (0.5% bovine serum albumin and 0.1% Tween 20 in PBS) were added and incubated overnight at 4 °C. After 3 washes in washing buffer, fluorescent-labeled secondary antibody diluted in washing buffer was added and incubated for 45 min at room temperature. Slides were washed at least six times in washing buffer, any remaining washing buffer was removed, and cells were mounted with hard set mounting media containing 4′,6-diamidino-2-phenylindole (Vector-Shield). Primary antibodies used were CSB H-300 rabbit (diluted 1: 500) and NEIL1 S-17 goat (diluted 1:75) (Santa Cruz); secondary antibodies were Alexa Flour 488 donkey anti-goat and Alexa Fluor 594 donkey anti-rabbit. Antibodies were checked for cross-reaction and bleed-through between the two channels by using one primary and both secondary antibodies. No cross-reaction or bleed-through was detected. Background from the secondary was checked using no primary and both secondary. Pictures were acquired on a Nikon Eclipse TE-2000e confocal microscope using the 60× objective lens, 0.2-μm z-stacks throughout the cells with the Volocity software. Quantification was done using Volocity software, selecting the more intense areas and only including anything bigger than one voxel as follows; signals with intensity four or more S.D. above the mean intensity were selected for both NEIL1 and CSB. Anything with a volume less than 1.5 voxel for both NEIL1 and CSB were excluded. Co-localization was measured as overlap between the selected CSB and NEIL1 signals that was bigger than 1.5 voxels. An average of three experiments (total of 90 cells counted) is presented together with representative pictures. For the images, low level intensity signal was removed, and the contrast was enhanced in Volocity software. Elevated Levels of FapyGua, FapyAde, and 8-OH-Gua in DNA from CSB Knock-out Mouse Tissues-It has been proposed that oxidative DNA damage accumulates in CSB-deficient cells and that this plays a role in the pathophysiology of CS, especially in the dysfunction of the central nervous system. However, previous studies either measured 8-OH-Gua specifically or sites sensitive to E. coli Fpg, which recognizes 8-OH-Gua, FapyGua, and FapyAde collectively (35Boiteux S. Gajewski E. Laval J. Dizdaroglu M. Biochemistry. 1992; 31: 106-110Crossref PubMed Scopus (571) Google Scholar). Here, the endogenous levels of 8-OH-Gua, FapyGua, and FapyAde were measured in liver, brain, and kidney genomic DNA and in liver mtDNA from wt or csb-/- mice by GC/MS. The results show that 8-OH-Gua is present at a significantly higher level in genomic DNA from brain and kidney of csb-/- than wt mice but not in liver DNA (Fig. 1A). FapyGua levels are also significantly higher in brain and kidney from csb-/- mice (Fig. 1B). FapyAde levels, on the other hand, are ∼2-fold higher in all three organs of csb-/- mice (Fig. 1C). The level of the nucleoside counterpart of 8-OH-Gua, 8-OH-dG, was measured in liver nuclear DNA using LC/MS, and almost identical results were obtained with GC
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