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W2018264891 abstract "We have isolated UvrB-DNA complexes by capture of biotinylated damaged DNA substrates on streptavidin-coated magnetic beads. With this method the UvrB-DNA preincision complex remains stable even in the absence of ATP. For the binding of UvrC to the UvrB-DNA complex no cofactor is needed. The subsequent induction of 3′ incision does require ATP binding by UvrB but not hydrolysis. This ATP binding induces a conformational change in the DNA, resulting in the appearance of the DNase I-hypersensitive site at the 5′ side of the damage. In contrast, the 5′ incision is not dependent on ATP binding because it occurs with the same efficiency with ADP. We show with competition experiments that both incision reactions are induced by the binding of the same UvrC molecule. A DNA substrate containing damage close to the 5′ end of the damaged strand is specifically bound by UvrB in the absence of UvrA and ATP (Moolenaar, G. F., Monaco, V., van der Marel, G. A., van Boom, J. H., Visse, R., and Goosen, N. (2000) J. Biol. Chem. 275, 8038–8043). To initiate the formation of an active UvrBC-DNA incision complex, however, UvrB first needs to hydrolyze ATP, and subsequently a new ATP molecule must be bound. Implications of these findings for the mechanism of the UvrA-mediated formation of the UvrB-DNA preincision complex will be discussed. We have isolated UvrB-DNA complexes by capture of biotinylated damaged DNA substrates on streptavidin-coated magnetic beads. With this method the UvrB-DNA preincision complex remains stable even in the absence of ATP. For the binding of UvrC to the UvrB-DNA complex no cofactor is needed. The subsequent induction of 3′ incision does require ATP binding by UvrB but not hydrolysis. This ATP binding induces a conformational change in the DNA, resulting in the appearance of the DNase I-hypersensitive site at the 5′ side of the damage. In contrast, the 5′ incision is not dependent on ATP binding because it occurs with the same efficiency with ADP. We show with competition experiments that both incision reactions are induced by the binding of the same UvrC molecule. A DNA substrate containing damage close to the 5′ end of the damaged strand is specifically bound by UvrB in the absence of UvrA and ATP (Moolenaar, G. F., Monaco, V., van der Marel, G. A., van Boom, J. H., Visse, R., and Goosen, N. (2000) J. Biol. Chem. 275, 8038–8043). To initiate the formation of an active UvrBC-DNA incision complex, however, UvrB first needs to hydrolyze ATP, and subsequently a new ATP molecule must be bound. Implications of these findings for the mechanism of the UvrA-mediated formation of the UvrB-DNA preincision complex will be discussed. adenosine 5′-O-(thiotriphosphate) Nucleotide excision repair in Escherichia coli is initiated by the binding of the UvrA2B complex to DNA containing damage. Following this, UvrB is loaded onto the site of the damage, and the UvrA protein is released. The resulting UvrB-DNA preincision complex is bound by the UvrC protein, leading to incision of the DNA at the 4th or 5th phosphodiester bond on the 3′ side of the damage. This 3′ incision is immediately followed by hydrolysis of the 8th phosphodiester bond at the 5′ side of the damage. The repair reaction is completed by the action of the UvrD, polymerase I, and ligase proteins, which replace the damaged oligo with a newly synthesized strand (for reviews see Refs. 1.Sancar A. Annu. Rev. Biochem. 1996; 65: 43-81Crossref PubMed Scopus (960) Google Scholar and 2.Goosen N. Moolenaar G.F. Visse R. van de Putte P. Eckstein F. Lilley D.M.J. Nucleic Acids and Molecular Biology: DNA Repair. Springer Verlag, Berlin1998: 103-123Google Scholar).ATP binding and hydrolysis play important roles throughout the repair reaction. The function of this cofactor is quite complex, which is illustrated by the presence of five ATP-binding sites in a single UvrA2B complex, two in each UvrA subunit and one in UvrB. The dimerization of UvrA is stimulated by ATP binding but not hydrolysis (3.Mazur S.J. Grossman L. Biochemistry. 1991; 30: 4432-4443Crossref PubMed Scopus (83) Google Scholar). The formation of an (active) UvrA2B complex in solution requires the hydrolysis of ATP by UvrA (4.Orren D.K. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5237-5241Crossref PubMed Scopus (167) Google Scholar). The ATP hydrolysis by UvrA is also an important factor in discriminating between damaged and nondamaged DNA (5.Oh E.Y. Grossman L. Nucleic Acids Res. 1986; 14: 16067-16071Crossref Scopus (46) Google Scholar). In solution the UvrB protein on its own does not hydrolyze ATP, but as part of the UvrA2B complex it displays a DNA damage-dependent ATPase activity (6.Seeley T.W. Grossman L. J. Biol. Chem. 1990; 265: 7158-7165Abstract Full Text PDF PubMed Google Scholar). This ATPase activity is associated with a limited DNA unwinding activity (7.Oh E.Y. Grossman L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3638-3642Crossref PubMed Scopus (96) Google Scholar), which was shown to be important for loading the UvrB protein onto the site of the damage (8.Moolenaar G.F. Visse R. Ortiz-Buysse M. Goosen N. van de Putte P. J. Mol. Biol. 1994; 240: 294-307Crossref PubMed Scopus (67) Google Scholar). Finally it has been shown that binding of ATP to the UvrBC-DNA complex is important for incision (9.Orren D.K. Sancar A. J. Biol. Chem. 1990; 265: 15796-15803Abstract Full Text PDF PubMed Google Scholar). In the latter experiments incision was monitored by the conversion of UV-irradiated supercoiled DNA substrate to the relaxed form, and therefore it could not be determined whether ATP binding is needed for 3′ incision alone or for both incision reactions.In this paper we take a closer look at the function of ATP binding and hydrolysis in formation of the UvrB-DNA preincision complex and formation of the UvrBC-DNA incision complex and in the two incision reactions. For this purpose we have constructed biotinylated damaged DNA substrates, which are used to capture repair intermediates on streptavidin-coated magnetic beads.DISCUSSIONOn a double-stranded damaged DNA substrate the loading of UvrB onto the site of the damage requires the action of UvrA and ATP. Several observations have suggested that the resulting UvrB-DNA preincision complex is stable in the presence of ATP only: (i) Isolation of UvrB-DNA complexes by column chromatography at room temperature was only possible if the chromatography buffers contained ATP (9.Orren D.K. Sancar A. J. Biol. Chem. 1990; 265: 15796-15803Abstract Full Text PDF PubMed Google Scholar). (ii) Separation of preincision complexes by gel retardation results in a much higher yield when ATP is included in the gel and the electrophoresis buffer (10.Visse R. de Ruijter M. Moolenaar G.F. van de Putte P. J. Biol. Chem. 1992; 267: 4820-4827Abstract Full Text PDF Google Scholar, 17.Moolenaar G.F. Monaco V. van de Marel G.A. van Boom J.H. Visse R. Goosen N. J. Biol. Chem. 2000; 275: 8038-8043Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). In this paper we show that a stable UvrB-DNA complex can be isolated in the absence of ATP by capture of a biotinylated damaged DNA substrate on streptavidin-coated magnetic beads. The complex survived multiple washes in buffer without ATP even at room temperature. Apparently the stability of the UvrB-DNA complex is highly dependent on the method used to separate it from the other components of the reaction mixture.Binding of ATP to the UvrB-DNA complex induces a conformational change in the DNA as was shown by DNase I footprinting. In the presence of ATP the UvrB-DNA complex shows a DNase I-hypersensitive site at the 5′ side of the damage. This site is generally believed to be characteristic for the formation of the preincision complex. The DNase I-hypersensitive site is also apparent in the presence of ATPγS but not with ADP, indicating that it is the ATP binding and not the hydrolysis that induces the specific DNA conformation. We will refer to the UvrB-DNA complex prior to the binding of ATP as the pro-preincision complex and with bound ATP as the preincision complex. The appearance of the DNase I-hypersensitive site fully correlates with the induction of 3′ incision after addition of UvrC to the isolated complexes; efficient incision occurs in the presence of ATP and ATPγS, but not with ADP or without cofactor. The binding of UvrC to the UvrB-DNA complex does not require a cofactor, suggesting that the ATP-induced conformational change is needed for the 3′ incision itself. The ATP-induced conformational change is not required for the 5′ incision. Although on a 3′ prenicked substrate ATP and ATPγS still specifically induce the DNase I-hypersensitive site, the 5′ incision is as efficient with ADP, which does not give this conformational change. The 5′ incision can even take place in the absence of cofactor, albeit at a very low level. Strikingly, addition of UvrC to isolated UvrB-DNA complexes formed on a 3′ prenicked substrate resulted in a very efficient 5′ incision at 0 °C, even after incubation for only 3 min. Apparently the binding of UvrC to the preincised complex directly docks the 5′ incision position into the catalytic site of the protein. The 3′ incision event, in contrast, appears to be much more difficult to achieve. In the accompanying paper (17.Moolenaar G.F. Monaco V. van de Marel G.A. van Boom J.H. Visse R. Goosen N. J. Biol. Chem. 2000; 275: 8038-8043Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) we have shown that in a UvrB-DNA complex without ATP the DNA region of the 3′ incision is under torsional stress, resulting in the instability of this pro-preincision complex as discussed above. This deformation of the DNA is important for the eventual 3′ incision, because relaxation of the DNA by introduction of a single strand nick opposite the 3′ site completely abolishes incision (17.Moolenaar G.F. Monaco V. van de Marel G.A. van Boom J.H. Visse R. Goosen N. J. Biol. Chem. 2000; 275: 8038-8043Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Subsequent binding of ATP to the pro-preincision complex not only induces the DNase I-hypersensitive site as we show here, but it also seems to release or compensate for the torsional stress in the 3′ region, because it stabilizes the complex in a retardation gel (17.Moolenaar G.F. Monaco V. van de Marel G.A. van Boom J.H. Visse R. Goosen N. J. Biol. Chem. 2000; 275: 8038-8043Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). These observations indicate that 3′ incision requires two consecutive conformational changes in the 3′ region of the DNA, the first one made during formation of the pro-preincision complex and the second one because of subsequent ATP binding. Moreover the 3′ incision appears also to require thermal energy, because addition of UvrC to preformed preincision complexes does not give any 3′ incision at 0 °C. Taken together, the exposure of the 3′ incision site to the catalytic residues seems to need a very specific protein-DNA conformation in which the DNA helix is likely to be considerably distorted. Recently we have shown that the UvrC protein contains two catalytic sites, one for the 3′ incision and one for the 5′ incision (21.Verhoeven E.E.A. van Kesteren M. Moolenaar G.F. Visse R. Goosen N. J. Biol. Chem. 2000; 275 (article number 806636)Google Scholar). From the results in this paper, it is clear that both incisions are made by the same UvrC molecule. The competition experiments indicate that the coiled-coil interaction between the C-terminal domain of UvrB and the homologous domain of UvrC is maintained after the 3′ incision has occurred, even though it has been shown that it is not essential for the 5′ incision (20.Moolenaar G.F. Franken K.L.M.C. Dijkstra D.M. Thomas-Oates J.E. Visse R. van de Putte P. Goosen N. J. Biol. Chem. 1995; 270: 30508-30515Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar).Damage recognition by the UvrB protein per se does not require ATP (17.Moolenaar G.F. Monaco V. van de Marel G.A. van Boom J.H. Visse R. Goosen N. J. Biol. Chem. 2000; 275: 8038-8043Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), which is why UvrB can specifically bind to the site of the damage in substrate G10 in the absence of cofactor. Before incision can occur, however, the UvrB-DNA complex on this substrate first needs to hydrolyze ATP, and then a new ATP molecule must be bound. These two ATP-dependent reactions are most likely required to induce the two consecutive conformational changes associated with formation of the pro-preincision complex and the preincision complex, respectively, as discussed above. On a double-stranded DNA substrate, ATP hydrolysis by UvrB is also needed in a prior step to trigger the DNA helicase activity of the UvrA2B complex, which is required for the loading of UvrB onto the damaged site (6.Seeley T.W. Grossman L. J. Biol. Chem. 1990; 265: 7158-7165Abstract Full Text PDF PubMed Google Scholar, 7.Oh E.Y. Grossman L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3638-3642Crossref PubMed Scopus (96) Google Scholar, 8.Moolenaar G.F. Visse R. Ortiz-Buysse M. Goosen N. van de Putte P. J. Mol. Biol. 1994; 240: 294-307Crossref PubMed Scopus (67) Google Scholar). In the accompanying paper (17.Moolenaar G.F. Monaco V. van de Marel G.A. van Boom J.H. Visse R. Goosen N. J. Biol. Chem. 2000; 275: 8038-8043Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) we have shown that this helicase activity presumably unwinds the DNA at the 5′ side of the damage, thereby allowing UvrB access to the damage.Taken together we come to a model in which UvrB hydrolyzes multiple ATP molecules during the repair reaction (Fig.8). First ATP hydrolysis by the UvrA2B complex is needed for opening up the DNA helix to bring UvrB close to the damage. Next the UvrB protein binds to this damaged site, and in the resulting UvrB·DNA complex a second round of ATP hydrolysis is triggered, thereby inducing the conformational changes that lead to formation of the relatively unstable pro-preincision complex. The experiments with substrate G10 have shown that this ATP hydrolysis can occur in the absence of UvrA. Therefore UvrA might be released from the complex during formation of the initial UvrB·DNA complex, although we cannot exclude the possibility that this dissociation occurs at a later stage. The binding of ATP to the pro-preincision complex induces formation of the preincision complex, which after binding of UvrC can be incised at the 3′ site. Finally, for the 5′ incision no further ATP binding or hydrolysis is needed. We have shown in this paper that the UvrC protein is capable of binding to all three UvrB-DNA intermediate complexes. In the normal chain of events the UvrB·DNA complex formed after loading of UvrB, and the pro-preincision complex are expected to be very short-lived. Thereforein vivo UvrC will most probably bind when the preincision complex is formed. Nucleotide excision repair in Escherichia coli is initiated by the binding of the UvrA2B complex to DNA containing damage. Following this, UvrB is loaded onto the site of the damage, and the UvrA protein is released. The resulting UvrB-DNA preincision complex is bound by the UvrC protein, leading to incision of the DNA at the 4th or 5th phosphodiester bond on the 3′ side of the damage. This 3′ incision is immediately followed by hydrolysis of the 8th phosphodiester bond at the 5′ side of the damage. The repair reaction is completed by the action of the UvrD, polymerase I, and ligase proteins, which replace the damaged oligo with a newly synthesized strand (for reviews see Refs. 1.Sancar A. Annu. Rev. Biochem. 1996; 65: 43-81Crossref PubMed Scopus (960) Google Scholar and 2.Goosen N. Moolenaar G.F. Visse R. van de Putte P. Eckstein F. Lilley D.M.J. Nucleic Acids and Molecular Biology: DNA Repair. Springer Verlag, Berlin1998: 103-123Google Scholar). ATP binding and hydrolysis play important roles throughout the repair reaction. The function of this cofactor is quite complex, which is illustrated by the presence of five ATP-binding sites in a single UvrA2B complex, two in each UvrA subunit and one in UvrB. The dimerization of UvrA is stimulated by ATP binding but not hydrolysis (3.Mazur S.J. Grossman L. Biochemistry. 1991; 30: 4432-4443Crossref PubMed Scopus (83) Google Scholar). The formation of an (active) UvrA2B complex in solution requires the hydrolysis of ATP by UvrA (4.Orren D.K. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5237-5241Crossref PubMed Scopus (167) Google Scholar). The ATP hydrolysis by UvrA is also an important factor in discriminating between damaged and nondamaged DNA (5.Oh E.Y. Grossman L. Nucleic Acids Res. 1986; 14: 16067-16071Crossref Scopus (46) Google Scholar). In solution the UvrB protein on its own does not hydrolyze ATP, but as part of the UvrA2B complex it displays a DNA damage-dependent ATPase activity (6.Seeley T.W. Grossman L. J. Biol. Chem. 1990; 265: 7158-7165Abstract Full Text PDF PubMed Google Scholar). This ATPase activity is associated with a limited DNA unwinding activity (7.Oh E.Y. Grossman L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3638-3642Crossref PubMed Scopus (96) Google Scholar), which was shown to be important for loading the UvrB protein onto the site of the damage (8.Moolenaar G.F. Visse R. Ortiz-Buysse M. Goosen N. van de Putte P. J. Mol. Biol. 1994; 240: 294-307Crossref PubMed Scopus (67) Google Scholar). Finally it has been shown that binding of ATP to the UvrBC-DNA complex is important for incision (9.Orren D.K. Sancar A. J. Biol. Chem. 1990; 265: 15796-15803Abstract Full Text PDF PubMed Google Scholar). In the latter experiments incision was monitored by the conversion of UV-irradiated supercoiled DNA substrate to the relaxed form, and therefore it could not be determined whether ATP binding is needed for 3′ incision alone or for both incision reactions. In this paper we take a closer look at the function of ATP binding and hydrolysis in formation of the UvrB-DNA preincision complex and formation of the UvrBC-DNA incision complex and in the two incision reactions. For this purpose we have constructed biotinylated damaged DNA substrates, which are used to capture repair intermediates on streptavidin-coated magnetic beads. DISCUSSIONOn a double-stranded damaged DNA substrate the loading of UvrB onto the site of the damage requires the action of UvrA and ATP. Several observations have suggested that the resulting UvrB-DNA preincision complex is stable in the presence of ATP only: (i) Isolation of UvrB-DNA complexes by column chromatography at room temperature was only possible if the chromatography buffers contained ATP (9.Orren D.K. Sancar A. J. Biol. Chem. 1990; 265: 15796-15803Abstract Full Text PDF PubMed Google Scholar). (ii) Separation of preincision complexes by gel retardation results in a much higher yield when ATP is included in the gel and the electrophoresis buffer (10.Visse R. de Ruijter M. Moolenaar G.F. van de Putte P. J. Biol. Chem. 1992; 267: 4820-4827Abstract Full Text PDF Google Scholar, 17.Moolenaar G.F. Monaco V. van de Marel G.A. van Boom J.H. Visse R. Goosen N. J. Biol. Chem. 2000; 275: 8038-8043Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). In this paper we show that a stable UvrB-DNA complex can be isolated in the absence of ATP by capture of a biotinylated damaged DNA substrate on streptavidin-coated magnetic beads. The complex survived multiple washes in buffer without ATP even at room temperature. Apparently the stability of the UvrB-DNA complex is highly dependent on the method used to separate it from the other components of the reaction mixture.Binding of ATP to the UvrB-DNA complex induces a conformational change in the DNA as was shown by DNase I footprinting. In the presence of ATP the UvrB-DNA complex shows a DNase I-hypersensitive site at the 5′ side of the damage. This site is generally believed to be characteristic for the formation of the preincision complex. The DNase I-hypersensitive site is also apparent in the presence of ATPγS but not with ADP, indicating that it is the ATP binding and not the hydrolysis that induces the specific DNA conformation. We will refer to the UvrB-DNA complex prior to the binding of ATP as the pro-preincision complex and with bound ATP as the preincision complex. The appearance of the DNase I-hypersensitive site fully correlates with the induction of 3′ incision after addition of UvrC to the isolated complexes; efficient incision occurs in the presence of ATP and ATPγS, but not with ADP or without cofactor. The binding of UvrC to the UvrB-DNA complex does not require a cofactor, suggesting that the ATP-induced conformational change is needed for the 3′ incision itself. The ATP-induced conformational change is not required for the 5′ incision. Although on a 3′ prenicked substrate ATP and ATPγS still specifically induce the DNase I-hypersensitive site, the 5′ incision is as efficient with ADP, which does not give this conformational change. The 5′ incision can even take place in the absence of cofactor, albeit at a very low level. Strikingly, addition of UvrC to isolated UvrB-DNA complexes formed on a 3′ prenicked substrate resulted in a very efficient 5′ incision at 0 °C, even after incubation for only 3 min. Apparently the binding of UvrC to the preincised complex directly docks the 5′ incision position into the catalytic site of the protein. The 3′ incision event, in contrast, appears to be much more difficult to achieve. In the accompanying paper (17.Moolenaar G.F. Monaco V. van de Marel G.A. van Boom J.H. Visse R. Goosen N. J. Biol. Chem. 2000; 275: 8038-8043Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) we have shown that in a UvrB-DNA complex without ATP the DNA region of the 3′ incision is under torsional stress, resulting in the instability of this pro-preincision complex as discussed above. This deformation of the DNA is important for the eventual 3′ incision, because relaxation of the DNA by introduction of a single strand nick opposite the 3′ site completely abolishes incision (17.Moolenaar G.F. Monaco V. van de Marel G.A. van Boom J.H. Visse R. Goosen N. J. Biol. Chem. 2000; 275: 8038-8043Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Subsequent binding of ATP to the pro-preincision complex not only induces the DNase I-hypersensitive site as we show here, but it also seems to release or compensate for the torsional stress in the 3′ region, because it stabilizes the complex in a retardation gel (17.Moolenaar G.F. Monaco V. van de Marel G.A. van Boom J.H. Visse R. Goosen N. J. Biol. Chem. 2000; 275: 8038-8043Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). These observations indicate that 3′ incision requires two consecutive conformational changes in the 3′ region of the DNA, the first one made during formation of the pro-preincision complex and the second one because of subsequent ATP binding. Moreover the 3′ incision appears also to require thermal energy, because addition of UvrC to preformed preincision complexes does not give any 3′ incision at 0 °C. Taken together, the exposure of the 3′ incision site to the catalytic residues seems to need a very specific protein-DNA conformation in which the DNA helix is likely to be considerably distorted. Recently we have shown that the UvrC protein contains two catalytic sites, one for the 3′ incision and one for the 5′ incision (21.Verhoeven E.E.A. van Kesteren M. Moolenaar G.F. Visse R. Goosen N. J. Biol. Chem. 2000; 275 (article number 806636)Google Scholar). From the results in this paper, it is clear that both incisions are made by the same UvrC molecule. The competition experiments indicate that the coiled-coil interaction between the C-terminal domain of UvrB and the homologous domain of UvrC is maintained after the 3′ incision has occurred, even though it has been shown that it is not essential for the 5′ incision (20.Moolenaar G.F. Franken K.L.M.C. Dijkstra D.M. Thomas-Oates J.E. Visse R. van de Putte P. Goosen N. J. Biol. Chem. 1995; 270: 30508-30515Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar).Damage recognition by the UvrB protein per se does not require ATP (17.Moolenaar G.F. Monaco V. van de Marel G.A. van Boom J.H. Visse R. Goosen N. J. Biol. Chem. 2000; 275: 8038-8043Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), which is why UvrB can specifically bind to the site of the damage in substrate G10 in the absence of cofactor. Before incision can occur, however, the UvrB-DNA complex on this substrate first needs to hydrolyze ATP, and then a new ATP molecule must be bound. These two ATP-dependent reactions are most likely required to induce the two consecutive conformational changes associated with formation of the pro-preincision complex and the preincision complex, respectively, as discussed above. On a double-stranded DNA substrate, ATP hydrolysis by UvrB is also needed in a prior step to trigger the DNA helicase activity of the UvrA2B complex, which is required for the loading of UvrB onto the damaged site (6.Seeley T.W. Grossman L. J. Biol. Chem. 1990; 265: 7158-7165Abstract Full Text PDF PubMed Google Scholar, 7.Oh E.Y. Grossman L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3638-3642Crossref PubMed Scopus (96) Google Scholar, 8.Moolenaar G.F. Visse R. Ortiz-Buysse M. Goosen N. van de Putte P. J. Mol. Biol. 1994; 240: 294-307Crossref PubMed Scopus (67) Google Scholar). In the accompanying paper (17.Moolenaar G.F. Monaco V. van de Marel G.A. van Boom J.H. Visse R. Goosen N. J. Biol. Chem. 2000; 275: 8038-8043Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) we have shown that this helicase activity presumably unwinds the DNA at the 5′ side of the damage, thereby allowing UvrB access to the damage.Taken together we come to a model in which UvrB hydrolyzes multiple ATP molecules during the repair reaction (Fig.8). First ATP hydrolysis by the UvrA2B complex is needed for opening up the DNA helix to bring UvrB close to the damage. Next the UvrB protein binds to this damaged site, and in the resulting UvrB·DNA complex a second round of ATP hydrolysis is triggered, thereby inducing the conformational changes that lead to formation of the relatively unstable pro-preincision complex. The experiments with substrate G10 have shown that this ATP hydrolysis can occur in the absence of UvrA. Therefore UvrA might be released from the complex during formation of the initial UvrB·DNA complex, although we cannot exclude the possibility that this dissociation occurs at a later stage. The binding of ATP to the pro-preincision complex induces formation of the preincision complex, which after binding of UvrC can be incised at the 3′ site. Finally, for the 5′ incision no further ATP binding or hydrolysis is needed. We have shown in this paper that the UvrC protein is capable of binding to all three UvrB-DNA intermediate complexes. In the normal chain of events the UvrB·DNA complex formed after loading of UvrB, and the pro-preincision complex are expected to be very short-lived. Thereforein vivo UvrC will most probably bind when the preincision complex is formed. On a double-stranded damaged DNA substrate the loading of UvrB onto the site of the damage requires the action of UvrA and ATP. Several observations have suggested that the resulting UvrB-DNA preincision complex is stable in the presence of ATP only: (i) Isolation of UvrB-DNA complexes by column chromatography at room temperature was only possible if the chromatography buffers contained ATP (9.Orren D.K. Sancar A. J. Biol. Chem. 1990; 265: 15796-15803Abstract Full Text PDF PubMed Google Scholar). (ii) Separation of preincision complexes by gel retardation results in a much higher yield when ATP is included in the gel and the electrophoresis buffer (10.Visse R. de Ruijter M. Moolenaar G.F. van de Putte P. J. Biol. Chem. 1992; 267: 4820-4827Abstract Full Text PDF Google Scholar, 17.Moolenaar G.F. Monaco V. van de Marel G.A. van Boom J.H. Visse R. Goosen N. J. Biol. Chem. 2000; 275: 8038-8043Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). In this paper we show that a stable UvrB-DNA complex can be isolated in the absence of ATP by capture of a biotinylated damaged DNA substrate on streptavidin-coated magnetic beads. The complex survived multiple washes in buffer without ATP even at room temperature. Apparently the stability of the UvrB-DNA complex is highly dependent on the method used to separate it from the other components of the reaction mixture. Binding of ATP to the UvrB-DNA complex induces a conformational change in the DNA as was shown by DNase I footprinting. In the presence of ATP the UvrB-DNA complex shows a DNase I-hypersensitive site at the 5′ side of the damage. This site is generally believed to be characteristic for the formation of the preincision complex. The DNase I-hypersensitive site is also apparent in the presence of ATPγS but not with ADP, indicating that it is the ATP binding and not the hydrolysis that induces the specific DNA conformation. We will refer to the UvrB-DNA complex prior to the binding of ATP as the pro-preincision complex and with bound ATP as the preincision complex. The appearance of the DNase I-hypersensitive site fully correlates with the induction of 3′ incision after addition of UvrC to the isolated complexes; efficient incision occurs in the presence of ATP and ATPγS, but not with ADP or without cofactor. The binding of UvrC to the UvrB-DNA complex does not require a cofactor, suggesting that the ATP-induced conformational change is needed for the 3′ incision itself. The ATP-induced conformational change is not required for the 5′ incision. Although on a 3′ prenicked substrate ATP and ATPγS still specifically induce the DNase I-hypersensitive site, the 5′ incision is as efficient with ADP, which does not give this conformational change. The 5′ incision can even take place in the absence of cofactor, albeit at a very low level. Strikingly, addition of UvrC to isolated UvrB-DNA complexes formed on a 3′ prenicked substrate resulted in a very efficient 5′ incision at 0 °C, even after incubation for only 3 min. Apparently the binding of UvrC to the preincised complex directly docks the 5′ incision position into the catalytic site of the protein. The 3′ incision event, in contrast, appears to be much more difficult to achieve. In the accompanying paper (17.Moolenaar G.F. Monaco V. van de Marel G.A. van Boom J.H. Visse R. Goosen N. J. Biol. Chem. 2000; 275: 8038-8043Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) we have shown that in a UvrB-DNA complex without ATP the DNA region of the 3′ incision is under torsional stress, resulting in the instability of this pro-preincision complex as discussed above. This deformation of the DNA is important for the eventual 3′ incision, because relaxation of the DNA by introduction of a single strand nick opposite the 3′ site completely abolishes incision (17.Moolenaar G.F. Monaco V. van de Marel G.A. van Boom J.H. Visse R. Goosen N. J. Biol. Chem. 2000; 275: 8038-8043Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Subsequent binding of ATP to the pro-preincision complex not only induces the DNase I-hypersensitive site as we show here, but it also seems to release or compensate for the torsional stress in the 3′ region, because it stabilizes the complex in a retardation gel (17.Moolenaar G.F. Monaco V. van de Marel G.A. van Boom J.H. Visse R. Goosen N. J. Biol. Chem. 2000; 275: 8038-8043Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). These observations indicate that 3′ incision requires two consecutive conformational changes in the 3′ region of the DNA, the first one made during formation of the pro-preincision complex and the second one because of subsequent ATP binding. Moreover the 3′ incision appears also to require thermal energy, because addition of UvrC to preformed preincision complexes does not give any 3′ incision at 0 °C. Taken together, the exposure of the 3′ incision site to the catalytic residues seems to need a very specific protein-DNA conformation in which the DNA helix is likely to be considerably distorted. Recently we have shown that the UvrC protein contains two catalytic sites, one for the 3′ incision and one for the 5′ incision (21.Verhoeven E.E.A. van Kesteren M. Moolenaar G.F. Visse R. Goosen N. J. Biol. Chem. 2000; 275 (article number 806636)Google Scholar). From the results in this paper, it is clear that both incisions are made by the same UvrC molecule. The competition experiments indicate that the coiled-coil interaction between the C-terminal domain of UvrB and the homologous domain of UvrC is maintained after the 3′ incision has occurred, even though it has been shown that it is not essential for the 5′ incision (20.Moolenaar G.F. Franken K.L.M.C. Dijkstra D.M. Thomas-Oates J.E. Visse R. van de Putte P. Goosen N. J. Biol. Chem. 1995; 270: 30508-30515Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Damage recognition by the UvrB protein per se does not require ATP (17.Moolenaar G.F. Monaco V. van de Marel G.A. van Boom J.H. Visse R. Goosen N. J. Biol. Chem. 2000; 275: 8038-8043Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), which is why UvrB can specifically bind to the site of the damage in substrate G10 in the absence of cofactor. Before incision can occur, however, the UvrB-DNA complex on this substrate first needs to hydrolyze ATP, and then a new ATP molecule must be bound. These two ATP-dependent reactions are most likely required to induce the two consecutive conformational changes associated with formation of the pro-preincision complex and the preincision complex, respectively, as discussed above. On a double-stranded DNA substrate, ATP hydrolysis by UvrB is also needed in a prior step to trigger the DNA helicase activity of the UvrA2B complex, which is required for the loading of UvrB onto the damaged site (6.Seeley T.W. Grossman L. J. Biol. Chem. 1990; 265: 7158-7165Abstract Full Text PDF PubMed Google Scholar, 7.Oh E.Y. Grossman L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3638-3642Crossref PubMed Scopus (96) Google Scholar, 8.Moolenaar G.F. Visse R. Ortiz-Buysse M. Goosen N. van de Putte P. J. Mol. Biol. 1994; 240: 294-307Crossref PubMed Scopus (67) Google Scholar). In the accompanying paper (17.Moolenaar G.F. Monaco V. van de Marel G.A. van Boom J.H. Visse R. Goosen N. J. Biol. Chem. 2000; 275: 8038-8043Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) we have shown that this helicase activity presumably unwinds the DNA at the 5′ side of the damage, thereby allowing UvrB access to the damage. Taken together we come to a model in which UvrB hydrolyzes multiple ATP molecules during the repair reaction (Fig.8). First ATP hydrolysis by the UvrA2B complex is needed for opening up the DNA helix to bring UvrB close to the damage. Next the UvrB protein binds to this damaged site, and in the resulting UvrB·DNA complex a second round of ATP hydrolysis is triggered, thereby inducing the conformational changes that lead to formation of the relatively unstable pro-preincision complex. The experiments with substrate G10 have shown that this ATP hydrolysis can occur in the absence of UvrA. Therefore UvrA might be released from the complex during formation of the initial UvrB·DNA complex, although we cannot exclude the possibility that this dissociation occurs at a later stage. The binding of ATP to the pro-preincision complex induces formation of the preincision complex, which after binding of UvrC can be incised at the 3′ site. Finally, for the 5′ incision no further ATP binding or hydrolysis is needed. We have shown in this paper that the UvrC protein is capable of binding to all three UvrB-DNA intermediate complexes. In the normal chain of events the UvrB·DNA complex formed after loading of UvrB, and the pro-preincision complex are expected to be very short-lived. Thereforein vivo UvrC will most probably bind when the preincision complex is formed." @default.
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W2018264891 title "The Role of ATP Binding and Hydrolysis by UvrB during Nucleotide Excision Repair" @default.
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