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• W1995374106 abstract "The current model for base excision repair (BER) involves two general sub-pathways termed single-nucleotide BER and long patch BER that are distinguished by their repair patch sizes and the enzymes/co-factors involved. Both sub-pathways involve a series of sequential steps from initiation to completion of repair. The BER sub-pathways are designed to sequester the various intermediates, passing them along from one step to the next without allowing these toxic molecules to trigger cell cycle arrest, necrotic cell death, or apoptosis. Although a variety of DNA-protein and protein-protein interactions are known for the BER intermediates and enzymes/co-factors, the molecular mechanisms accounting for step-to-step coordination are not well understood. In the present study we designed an in vitro assay to explore the question of whether there is a channeling or “hand-off” of the repair intermediates during BER in vitro. The results show that when BER enzymes are pre-bound to the initial single-nucleotide BER intermediate, the DNA is channeled from apurinic/apyrimidinic endonuclease 1 to DNA polymerase β and then to DNA ligase. In the long patch BER subpathway, where the 5′-end of the incised strand is blocked, the intermediate after DNA polymerase β gap filling is not channeled to the subsequent enzyme, flap endonuclease 1. Instead, flap endonuclease 1 must recognize and bind to the intermediate in competition with other molecules. The current model for base excision repair (BER) involves two general sub-pathways termed single-nucleotide BER and long patch BER that are distinguished by their repair patch sizes and the enzymes/co-factors involved. Both sub-pathways involve a series of sequential steps from initiation to completion of repair. The BER sub-pathways are designed to sequester the various intermediates, passing them along from one step to the next without allowing these toxic molecules to trigger cell cycle arrest, necrotic cell death, or apoptosis. Although a variety of DNA-protein and protein-protein interactions are known for the BER intermediates and enzymes/co-factors, the molecular mechanisms accounting for step-to-step coordination are not well understood. In the present study we designed an in vitro assay to explore the question of whether there is a channeling or “hand-off” of the repair intermediates during BER in vitro. The results show that when BER enzymes are pre-bound to the initial single-nucleotide BER intermediate, the DNA is channeled from apurinic/apyrimidinic endonuclease 1 to DNA polymerase β and then to DNA ligase. In the long patch BER subpathway, where the 5′-end of the incised strand is blocked, the intermediate after DNA polymerase β gap filling is not channeled to the subsequent enzyme, flap endonuclease 1. Instead, flap endonuclease 1 must recognize and bind to the intermediate in competition with other molecules. Mammalian genomic DNA is constantly exposed to a variety of physical and chemical agents, including ultraviolet light and ionization radiation, alkylating molecules, and endogenous reactive oxygen species that accumulate in cells due to environmental stress and natural metabolic processes (1Lindahl T. Annu. Rev. Biochem. 1982; 51: 61-87Crossref PubMed Scopus (696) Google Scholar, 2Lindahl T. Nature. 1993; 362: 709-715Crossref PubMed Scopus (4372) Google Scholar, 3Loeb L.A. Preston B.D. Annu. Rev. Genet. 1986; 20: 201-230Crossref PubMed Scopus (859) Google Scholar). However, cells have evolved several specific pathways to remove such damage and maintain integrity of the genome. In mammalian cells the primary means for correcting discrete small DNA base lesions is base excision repair (BER) 2The abbreviations used are: BERbase excision repairSN BERsingle-nucleotide BERLP BERlong-patch BERdRPdeoxyribose phosphateUDGuracil-DNA glycosylaseAPapurinic/apyrimidinicAPE1AP endonuclease 1pol βDNA polymerase βTHFtetrahydrofuranntnucleotide. (4Kubota Y. Nash R.A. Klungland A. Schär P. Barnes D.E. Lindahl T. EMBO J. 1996; 15: 6662-6670Crossref PubMed Scopus (692) Google Scholar, 5Wilson S.H. Mutat. Res. 1998; 407: 203-215Crossref PubMed Scopus (265) Google Scholar, 6Wilson 3rd, D.M. Thompson L.H. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 12754-12757Crossref PubMed Scopus (216) Google Scholar, 7Lindahl T. Wood R.D. Science. 1999; 286: 1897-1905Crossref PubMed Scopus (1279) Google Scholar). The current and widely accepted working model for mammalian BER is that the process involves two sub-pathways that are differentiated by repair patch size and enzymes involved (8Frosina G. Fortini P. Rossi O. Carrozzino F. Raspaglio G. Cox L.S. Lane D.P. Abbondandolo A. Dogliotti E. J. Biol. Chem. 1996; 271: 9573-9578Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 9Klungland A. Lindahl T. EMBO J. 1997; 16: 3341-3348Crossref PubMed Scopus (665) Google Scholar, 10Fortini P. Pascucci B. Parlanti E. Sobol R.W. Wilson S.H. Dogliotti E. Biochemistry. 1998; 37: 3575-3580Crossref PubMed Scopus (200) Google Scholar, 11Biade S. Sobol R.W. Wilson S.H. Matsumoto Y. J. Biol. Chem. 1998; 273: 898-902Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). These sub-pathways are termed “single-nucleotide BER” (SN BER) and “long-patch BER” (LP BER). SN BER involves removal of a single damaged nucleotide that is replaced with an undamaged nucleotide through template-directed DNA synthesis to fill the single-nucleotide gap, whereas in LP BER two or more nucleotides are replaced (8Frosina G. Fortini P. Rossi O. Carrozzino F. Raspaglio G. Cox L.S. Lane D.P. Abbondandolo A. Dogliotti E. J. Biol. Chem. 1996; 271: 9573-9578Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 9Klungland A. Lindahl T. EMBO J. 1997; 16: 3341-3348Crossref PubMed Scopus (665) Google Scholar, 10Fortini P. Pascucci B. Parlanti E. Sobol R.W. Wilson S.H. Dogliotti E. Biochemistry. 1998; 37: 3575-3580Crossref PubMed Scopus (200) Google Scholar, 11Biade S. Sobol R.W. Wilson S.H. Matsumoto Y. J. Biol. Chem. 1998; 273: 898-902Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 12Singhal R.K. Prasad R. Wilson S.H. J. Biol. Chem. 1995; 270: 949-957Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 13Dianov G. Price A. Lindahl T. Mol. Cell. Biol. 1992; 12: 1605-1612Crossref PubMed Scopus (264) Google Scholar). Both of the BER sub-pathways are processed sequentially, one step to the next, and are initiated either by enzymatic removal of a damaged base or by spontaneous chemical hydrolysis of the glycosidic bond connecting the damaged base to the sugar phosphate backbone (1Lindahl T. Annu. Rev. Biochem. 1982; 51: 61-87Crossref PubMed Scopus (696) Google Scholar, 2Lindahl T. Nature. 1993; 362: 709-715Crossref PubMed Scopus (4372) Google Scholar, 14Mosbaugh D.W. Bennett S.E. Prog. Nucleic Acid Res. Mol. Biol. 1994; 48: 315-370Crossref PubMed Scopus (98) Google Scholar, 15Slupphaug G. Eftedal I. Kavli B. Bharati S. Helle N.M. Haug T. Levine D.W. Krokan H.E. Biochemistry. 1995; 34: 128-138Crossref PubMed Scopus (247) Google Scholar). The resulting apurinic/apyrimidinic (AP) site is processed by AP endonuclease 1 (APE1) that incises the phosphodiester backbone 5′ to the abasic site, leaving a single-nucleotide gap with 3′-hydroxyl and 5′-deoxyribose phosphate at the gap margins (16Doetsch P.W. Helland D.E. Haseltine W.A. Biochemistry. 1986; 25: 2212-2220Crossref PubMed Scopus (93) Google Scholar, 17Doetsch P.W. Cunningham R.P. Mutat. Res. 1990; 236: 173-201Crossref PubMed Scopus (328) Google Scholar). DNA polymerase β (pol β), a multifunctional enzyme consisting of the 8-kDa amino-terminal domain with deoxyribose phosphate (dRP) lyase activity (18Matsumoto Y. Kim K. Science. 1995; 269: 699-702Crossref PubMed Scopus (650) Google Scholar, 19Piersen C.E. Prasad R. Wilson S.H. Lloyd R.S. J. Biol. Chem. 1996; 271: 17811-17815Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) and the 31-kDa carboxyl-terminal domain with nucleotidyltransferase activity (20Casas-Finet J.R. Kumar A. Morris G. Wilson S.H. Karpel R.L. J. Biol. Chem. 1991; 266: 19618-19625Abstract Full Text PDF PubMed Google Scholar, 21Kumar A. Abbotts J. Karawya E.M. Wilson S.H. Biochemistry. 1990; 29: 7156-7159Crossref PubMed Scopus (89) Google Scholar, 22Kumar A. Widen S.G. Williams K.R. Kedar P. Karpel R.L. Wilson S.H. J. Biol. Chem. 1990; 265: 2124-2131Abstract Full Text PDF PubMed Google Scholar), then catalyzes template-guided gap filling and removal of the 5-dRP group to generate the substrate for the final BER step, DNA ligation (12Singhal R.K. Prasad R. Wilson S.H. J. Biol. Chem. 1995; 270: 949-957Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 13Dianov G. Price A. Lindahl T. Mol. Cell. Biol. 1992; 12: 1605-1612Crossref PubMed Scopus (264) Google Scholar, 18Matsumoto Y. Kim K. Science. 1995; 269: 699-702Crossref PubMed Scopus (650) Google Scholar, 19Piersen C.E. Prasad R. Wilson S.H. Lloyd R.S. J. Biol. Chem. 1996; 271: 17811-17815Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 23Dianov G. Lindahl T. Curr. Biol. 1994; 4: 1069-1076Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 24Sobol R.W. Horton J.K. Kühn R. Gu H. Singhal R.K. Prasad R. Rajewsky K. Wilson S.H. Nature. 1996; 379: 183-186Crossref PubMed Scopus (787) Google Scholar, 25Srivastava D.K. Berg B.J. Prasad R. Molina J.T. Beard W.A. Tomkinson A.E. Wilson S.H. J. Biol. Chem. 1998; 273: 21203-21209Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar, 26Prasad R. Beard W.A. Strauss P.R. Wilson S.H. J. Biol. Chem. 1998; 273: 15263-15270Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). The ligation step is completed either by DNA ligase I or XRCC1-DNA ligase III complex (27Prasad R. Singhal R.K. Srivastava D.K. Molina J.T. Tomkinson A.E. Wilson S.H. J. Biol. Chem. 1996; 271: 16000-16007Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 28Prigent C. Satoh M.S. Daly G. Barnes D.E. Lindahl T. Mol. Cell. Biol. 1994; 14: 310-317Crossref PubMed Scopus (129) Google Scholar, 29Caldecott K.W. McKeown C.K. Tucker J.D. Ljungquist S. Thompson L.H. Mol. Cell. Biol. 1994; 14: 68-76Crossref PubMed Google Scholar). base excision repair single-nucleotide BER long-patch BER deoxyribose phosphate uracil-DNA glycosylase apurinic/apyrimidinic AP endonuclease 1 DNA polymerase β tetrahydrofuran nucleotide. The BER repair system and many of the BER enzymes are conserved in bacteria to humans, and these repair pathways have been reconstituted using purified natural and recombinant enzymes from various organisms (13Dianov G. Price A. Lindahl T. Mol. Cell. Biol. 1992; 12: 1605-1612Crossref PubMed Scopus (264) Google Scholar, 23Dianov G. Lindahl T. Curr. Biol. 1994; 4: 1069-1076Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). From biochemical and structural studies, a common theme emerged that the individual steps in BER are sequentially ordered. For example, it is only after damaged base removal that the AP site-containing DNA strand is recognized by APE1. DNA polymerase β then processes the gapped intermediate, generating the substrate for DNA ligase. Thus, to prevent exposure of the intermediates to harmful nuclease activities, recombination events, or cell death signaling, it appears that the BER enzymes must coordinate with one another to efficiently receive the DNA substrates and pass the resulting DNA products along to the next enzyme in the sequence, just as a baton is passed from one individual to the next in a relay (30Mol C.D. Izumi T. Mitra S. Tainer J.A. Nature. 2000; 403: 451-456Crossref PubMed Scopus (615) Google Scholar, 31Parikh S.S. Mol C.D. Hosfield D.J. Tainer J.A. Curr. Opin. Struct. Biol. 1999; 9: 37-47Crossref PubMed Scopus (108) Google Scholar, 32Wilson S.H. Kunkel T.A. Nat. Struct. Biol. 2000; 7: 176-178Crossref PubMed Scopus (325) Google Scholar). In the present study we tested the hypothesis that DNA can be channeled from one step to the next during BER. Steps in a reconstituted SN BER system were examined including APE1 strand incision, DNA synthesis, and dRP removal mediated by pol β, and ligation was mediated either by DNA ligase I or XRCC1-DNA ligase III complex. This was accomplished by preloading the enzymes onto BER substrates and then conducting the repair incubation in the presence of a trap such that any free enzyme or enzymes released from the DNA substrates during repair could no longer participate in the reactions. In the LP BER sub-pathway, we examined channeling of the DNA substrate from the DNA synthesis step to the FEN1 cleavage step. Our results show that the BER intermediates of the SN BER sub-pathway could be channeled from APE1 to pol β and to DNA ligase. In contrast, in the LP BER sub-pathway, the DNA product after gap filling by pol β was not channeled to FEN1. High pressure liquid chromatography-purified oligodeoxynucleotides were from Oligos Etc., Inc. (Wilsonville, OR) and The Midland Certified Reagent Co. (Midland, TX). [α-32P]dCTP, [α-32P]cordycepin (3000 Ci/mmol), and [γ-32P]ATP (6000 Ci/mmol) were from PerkinElmer Life Sciences. Optikinase and terminal deoxynucleotidyltransferase were from United States Biochemical Corp. (Cleveland, OH) and Fermentas Inc. (Hanover, MD), respectively. Recombinant human pol β was overexpressed and purified as described previously (33Beard W.A. Wilson S.H. Methods Enzymol. 1995; 262: 98-107Crossref PubMed Scopus (160) Google Scholar). Human APE1, uracil-DNA glycosylase (UDG) with 84 amino acids deleted from the amino terminus, FEN1, DNA ligase I, and His-tagged DNA ligase III were purified as described previously (15Slupphaug G. Eftedal I. Kavli B. Bharati S. Helle N.M. Haug T. Levine D.W. Krokan H.E. Biochemistry. 1995; 34: 128-138Crossref PubMed Scopus (247) Google Scholar, 34Prasad R. Dianov G.L. Bohr V.A. Wilson S.H. J. Biol. Chem. 2000; 275: 4460-4466Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 35Strauss P.R. Beard W.A. Patterson T.A. Wilson S.H. J. Biol. Chem. 1997; 272: 1302-1307Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 36Wang Y.C. Burkhart W.A. Mackey Z.B. Moyer M.B. Ramos W. Husain I. Chen J. Besterman J.M. Tomkinson A.E. J. Biol. Chem. 1994; 269: 31923-31928Abstract Full Text PDF PubMed Google Scholar, 37Chen X. Pascal J. Vijayakumar S. Wilson G.M. Ellenberger T. Tomkinson A.E. Methods Enzymol. 2006; 409: 39-52Crossref PubMed Scopus (29) Google Scholar). The protein concentrations of enzyme samples specified below were measured by routine protein assays; the active fraction in each case corresponded to ∼50%. Preparation of the 3′- or 5′-end-labeled dRP lyase substrate was as described previously (26Prasad R. Beard W.A. Strauss P.R. Wilson S.H. J. Biol. Chem. 1998; 273: 15263-15270Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). For the APE1 incision activity, the 32P-labeled duplex DNA was pretreated with UDG to generate an AP site. For the preparation of the 5′-end-labeled substrate, dephosphorylated 15-mer oligonucleotide (5′-CTG CAG CTG ATG CGC-3′) was phosphorylated with Optikinase and [γ-32P]ATP. A 34-mer (5′-GTA CCC GGG GAT CCG TAC GGC GCA TCA GCT GCA G-3′) template was then annealed with 32P-labeled 15-mer upstream and 19-mer (5′-pUGTACGGATCCCCGGGTAC-3′) downstream oligonucleotides by heating the solution at 90 °C for 3 min and allowing the solution to slowly cool to 25 °C. In some cases the 19-mer oligonucleotide was also 3′-labeled with [α-32P]ddATP before annealing. The 32P-labeled duplex DNA (labeled either at the 5′-end, 3′-end, or both ends) was treated with UDG to generate the 5′-dRP-containing single-nucleotide gapped substrate. In some experiments a 35-mer duplex DNA was used with uracil opposite guanine at position 15 in one strand and with the 3′-end blocked with cordycepin (3′-deoxyadenosine). In those experiments DNA was pretreated with UDG and APE1 to generate a single-nucleotide-gapped substrate with 3′-OH and 5′-dRP flap at the gap margins, and [α-32P]dCTP was used as radiolabel. To measure the incision activity of APE1 in the presence of a DNA trap, a reaction mixture containing 50 mm HEPES, pH 7.5, 20 mm KCl, 2 mm dithiothreitol, 0.5 mm EDTA, and 10 nm UDG-pretreated 32P-labeled or 40 μm unlabeled DNA trap (a synthetic AP site-containing oligonucleotide DNA with tetrahydrofuran (THF), mimicking the AP site) and 30 nm APE1 was assembled on ice. The reaction was initiated by adding a mixture of 40 μm DNA trap, 5 mm MgCl2, or 10 nm UDG-pretreated 5′-end 32P-labeled DNA substrate, 5 mm MgCl2 and transferring the reaction mixture to 30 °C. In some experiments when both incision and gap-filling DNA synthesis reactions were performed simultaneously, the reactions were initiated by adding a mixture of 40 μm DNA trap, 25 μm dCTP, and 5 mm MgCl2 or 10 nm UDG-pretreated 5′-end 32P-labeled DNA substrate, 25 μm dCTP, and 5 mm MgCl2. In Fig. 5, APE1 and pol β concentrations were 30 and 40 nm, respectively. Samples were withdrawn at 10 s and mixed with gel-loading dye solution. The mixtures were then heated at 75 °C for 2 min, and DNA products were separated by electrophoresis in a 15% polyacrylamide gel containing 8 m urea in 89 mm Tris-HCl, 89 mm boric acid, and 2 mm EDTA, pH 8.8. Imaging and data analysis were performed with phosphorimaging and ImageQuant™ software. To measure the dRP lyase activity of pol β in the presence of a DNA trap, a reaction mixture containing 60 nm pol β, 50 mm HEPES, pH 7.5, 20 mm KCl, 2 mm dithiothreitol, 1 mm EDTA, and 20 nm 3′-end 32P-labeled DNA substrate or 10 μm unlabeled DNA trap was assembled on ice. The reaction was initiated by adding a mixture of 10 μm DNA trap or 20 nm 3′-end 32P-labeled DNA substrate and transferring the reaction mixture to 30 °C. Samples were withdrawn at 10 and 20 s, and the reaction products were stabilized by the addition of freshly prepared 20 mm NaBH4. Reaction mixtures then were put on ice and incubated 20 min. After this incubation an equal volume of gel-loading dye solution was added, and the mixtures were heated at 75 °C for 2 min. Reaction products were then separated by electrophoresis in a 15% polyacrylamide gel containing 8 m urea in 89 mm Tris-HCl, 89 mm boric acid, and 2 mm EDTA, pH 8.8. Imaging and data analysis were performed with phosphorimaging and ImageQuant™ software. To measure the gap-filling DNA synthesis activity of pol β in the presence of a DNA trap, reaction mixtures containing the same components as described for the dRP lyase assay were assembled with some exceptions; the substrate was 5′-end-labeled, instead of 3′-end-labeled, and 2 μm dCTP and 5 mm MgCl2 were added to initiate the reaction along with the DNA trap. After incubation, an equal volume of gel-loading dye was added, and the reaction mixtures were heated at 75 °C for 2 min. Reaction products were then separated by electrophoresis in a 15% polyacrylamide gel containing 8 m urea in 89 mm Tris-HCl, 89 mm boric acid, and 2 mm EDTA, pH 8.8. Imaging and data analysis were performed with phosphorimaging and ImageQuant™ software. To perform combined gap-filling DNA synthesis and ligation reactions in the presence of a DNA trap, a reaction mixture containing 60 nm pol β, 50 mm HEPES, pH 7.5, 20 mm KCl, 2 mm dithiothreitol, 0.5 mm EDTA, 20 nm UDG/APE1-pretreated gapped DNA, or 10 μm unlabeled DNA trap was assembled on ice. The reaction was initiated by transferring the reaction mixture to 30 °C and adding a mixture of 10 μm unlabeled DNA trap or 20 nm UDG/APE1-pretreated gapped DNA substrate, 2.6 μm [α-32P]dCTP, and 5 mm MgCl2. Samples were withdrawn at 10 s, and the reaction was terminated by the addition of an equal volume of gel-loading dye solution. After incubation at 75 °C for 2 min, the reaction products were separated by electrophoresis and analyzed as above. However, in those cases when gap-filling, dRP lyase, and ligation steps were performed in combination, the reaction mixtures were supplemented with 4 mm ATP, 0.5 mm NAD, and 200 nm DNA ligase I. To examine gap-filling DNA synthesis, FEN1 cleavage, and ligation reactions in the presence of a DNA trap, a reaction mixture containing 50 mm HEPES, pH 7.5, 20 mm KCl, 2 mm dithiothreitol, 1 mm EDTA, 20 nm APE1-pretreated THF-containing DNA or 10 μm unlabeled DNA trap, and 60 nm pol β and/or 25 nm FEN1 was assembled on ice. The reaction was initiated by adding a mixture of 10 μm unlabeled DNA trap or 20 nm APE1-pretreated THF-containing DNA substrate, 2.6 μm [α-32P]dCTP, 20 μm each dATP, dGTP, and TTP, and 5 mm MgCl2. When all three steps (gap-filling DNA synthesis, FEN1 cleavage, and ligation) were conducted in combination or in the same sample, a reaction mixture containing 60 nm pol β, 25 nm FEN1, and 200 nm DNA ligase I was incubated with DNA substrate or the trap as above. All incubations were at 30 °C. Samples were withdrawn at 10 s, and the reaction was terminated by the addition of an equal volume of gel-loading dye solution. After incubation at 75 °C for 2 min, the reaction products were separated by electrophoresis and analyzed as above. However, when gap-filling, FEN1 cleavage, and ligation steps were performed in combination, the reaction mixtures were also supplemented with 4 mm ATP and 200 nm DNA ligase I. Single-nucleotide gap filling reactions utilized the same UDG-processed nicked substrate that was prepared for dRP lyase assay reaction mixtures. In these present reactions, however, the primer strand was 5′-end 32P-labeled. Single turnover assays (E > S) were performed to measure insertion of dCTP (i.e. templating dG in the gap) using a KinTek Model RQF-3 rapid quench-flow system (Austin, TX). Unless noted otherwise, all concentrations refer to the final reaction concentrations after mixing. The final concentrations were 100 nm DNA, 500 nm pol β, 100 μm dCTP, 5 mm MgCl2, 50 mm HEPES-KOH, pH 7.5, 20 mm KCl, 0.5 mm EDTA, 1 mm dithiothreitol, 50 μg/ml bovine serum albumin, and 10% glycerol. After various time periods at 15 °C, reactions were quenched with 0.25 m EDTA. Quenched reactions were mixed with formamide dye, heated (5 min at 95 °C), and separated on a 20% polyacrylamide gel containing 8 m urea. Substrate and product bands were quantified after exposure to a phosphor screen using ImageQuant™ software. Time courses were fitted to a rising exponential, yielding the rate of single nucleotide insertion. The first-order rate constant is equivalent to the insertion rate (kpol). Steady-state dRP lyase reactions used the 3′-end 32P-labeled gapped DNA with a 5′-dRP group in the gap. The preparation of this substrate is described above. Reactions were initiated with pol β (50, 100, or 150 nm) and 500 nm labeled DNA substrate in 50 mm HEPES-KOH, pH 7.5, 20 mm KCl, 0.5 mm EDTA, 1 mm dithiothreitol, 50 μg/ml bovine serum albumin, and 10% glycerol. After various time periods at 15 °C, 10 μl of the reaction mixture was removed and quenched with an equal volume of freshly prepared 0.4 m NaBH4. Quenched reactions were mixed with formamide dye, heated, and resolved on 20% polyacrylamide gels containing 8 m urea. Bands were visualized by exposure to a phosphorimaging screen and analyzed using ImageQuant™ software. Single turnover dRP lyase reactions utilized the 3′-end 32P-labeled gapped DNA with a 5′-dRP group in the gap. The preparation of this substrate is described above. Time courses were performed using the chemical quench-flow system. In these assays, pol β and pretreated 3′-end 32P-labeled DNA were rapidly mixed for various times at 15 °C, and the reactions quenched by collection into tubes containing freshly prepared 0.4 m NaBH4. The final concentrations were 500 nm pol β, 100 nm DNA substrate, 50 mm HEPES-KOH, pH 7.5, 20 mm KCl, 0.5 mm EDTA, 1 mm dithiothreitol, 50 μg/ml bovine serum albumin, and 10% glycerol. Quench flow parameters were input manually to obtain the desired time points when quenching occurred in the collection tubes, thereby correcting for the time that each reaction mixture spent in the exit loop. This was necessary due to the volatile nature of the high concentration of NaBH4. The DNA substrate and product were ethanol-precipitated in the presence of linear polyacrylamide carrier (10–15 μg) and re-suspended in formamide gel loading buffer. After brief heating (5 min at 95 °C), the DNA was resolved on a 20% polyacrylamide gel containing 8 m urea. The dried gel was exposed to a phosphorimaging screen and quantified using ImageQuant™ software. Time courses were fitted to a rising exponential with a base-line term. Biochemical and structural studies of BER enzymes suggested that BER intermediates could be passed sequentially from one enzyme to the next in a coordinated fashion, in contrast to a model where all substrates and products are in equilibrium with free enzymes. To examine the hypothesis that the product of one step can be channeled as substrate to the next enzyme in the SN BER pathway, we studied AP site incision, gap filling, removal of the 5′-dRP group, and ligation. These steps are mediated by APE1, pol β, and DNA ligase I, respectively, and were studied here either individually or in combination after preloading the enzyme(s) onto DNA and initiating the reactions in the presence of a trap. The design for these “single turnover” repair experiments is summarized in Scheme 1. Either an individual BER enzyme or a mixture of enzymes was first preincubated with the respective substrate DNA, and then the enzyme-substrate complex was mixed with a DNA trap plus an initiator for the reaction(s). After a brief incubation, products were recovered by gel electrophoresis and quantified. Scheme 1 illustrates the different types of incubations used; type 1 involved individual BER enzymes and steps as shown, and types 2 and 3 involved mixtures of BER enzymes and more than one reaction product. In the preincubations, the substrate DNA concentrations were in the range of the dissociation constants for the respective enzymes (10–20 nm). The reaction products observed in the presence of trap resulted from turnover of enzyme that was bound during the preincubation. The incubations were for 10 s, the shortest period we could manage using manual methods but much longer than the catalytic rate constants for each enzyme. In LP BER, the DNA synthesis reaction may occur as a series of single-nucleotide gap filling steps in conjunction with FEN1 5′-tailoring (38Liu Y. Beard W.A. Shock D.D. Prasad R. Hou E.W. Wilson S.H. J. Biol. Chem. 2005; 280: 3665-3674Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Accordingly, we also examined whether FEN1 can preload onto its substrate, conduct flap removal, and then pass the product to next step, gap-filling DNA synthesis. To study the APE1-mediated BER step, the reaction mixtures were assembled and incubated as shown in Scheme 1. The 32P-labeled DNA containing a lone uracil residue at position 15 was pretreated with UDG to generate an AP site-containing substrate. A 34-bp unlabeled DNA containing a synthetic AP site (THF) was used to trap any unbound APE1 in the reaction mixture. The reaction mixture was assembled on ice and initiated by transferring the reaction mixture to 30 °C and adding a mixture of MgCl2 and ∼4000-fold excess DNA trap. In another set of control reaction mixtures, APE1 was preincubated with trap first, and the reaction was initiated as above. The results showed that APE1 prebound to the DNA substrate was able to incise the AP site (Fig. 1a); unbound APE1 in solution was quenched by the trap, as no increase in product formation was observed with a 20-s incubation (not shown). In contrast, when APE1 was first preincubated with DNA trap and then the reaction was initiated by adding a mixture of DNA substrate and MgCl2, very little product was observed. This indicated that the trap was able to capture almost all of APE1 in the reaction mixture before it could bind and incise DNA substrate (Fig. 1b). Under these conditions 22% of the substrate was converted to product. To study these two pol β-mediated BER steps, reaction mixtures were assembled and incubated for 10 and 20 s. To examine DNA synthesis, a 34-mer nicked DNA was first prepared by annealing 5′-end-labeled 15- and 19-mer oligonucleotides to a complementary 34-mer DNA strand. The 19-mer oligonucleotide contained a 5′-phosphorylated uracil nucleotide. This duplex DNA was pretreated with UDG, resulting in a single-nucleotide gapped DNA with 3′-OH and 5′-dRP groups at the gap margins, mimicking the APE1-incised BER intermediate. A 21-bp unlabeled gapped DNA was used to trap any unbound pol β in the reaction mixture. To examine use of this BER intermediate by pol β in the DNA synthesis step, the reaction mixture was assembled on ice, and the reaction was initiated by transferring the tubes to 30 °C and adding a mixture of dCTP, MgCl2, and a ∼500-fold excess of trap. In another set of reaction mixtures, pol β was preincubated with the trap first, and the reaction was initiated by adding a mixture of DNA substrate, dCTP, and MgCl2. The reaction mixtures were incubated at 30 °C, and samples were withdrawn for products analysis. The results showed that pol β pre-bound to the DNA substrate was able to incorporate one nucleotide (Fig. 2a, lane 2), i.e. incorporation of dCMP into DNA; unbound pol β in solution was trapped completely as no further increase in dCMP incorporation was observed with a 20-s incubation (Fig. 2a, lane 3). In contrast, when pol β was first preincubated with the trap and then the reaction was initiated by adding a mixture of DNA substrate, dCTP, and MgCl2, no incorporation of dCMP was observed. This indicated that the enzyme was productively bound during the preincubation and that the trap was able to capture all of the pol β in the reaction mixture (Fig. 2a, lanes 4 and 5). Under these conditions 25% of the" @default.
• W1995374106 created "2016-06-24" @default.
• W1995374106 creator A5004213173 @default.
• W1995374106 creator A5045523035 @default.
• W1995374106 creator A5062571628 @default.
• W1995374106 creator A5089715804 @default.
• W1995374106 date "2010-12-01" @default.
• W1995374106 modified "2023-10-13" @default.
• W1995374106 title "Substrate Channeling in Mammalian Base Excision Repair Pathways: Passing the Baton" @default.
• W1995374106 cites W1409078517 @default.
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