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Unraveling the Mechanistic Details of Topoisomerases
Structure 502
Andreas Bracher and F. Ulrich Hartl Department of Cellular Biochemistry Max Planck Institute of Biochemistry Am Klopferspitz 18 82152 Martinsried Germany
Huai, Q., Wang, H., Liu, Y., Kim, H.-Y., Toft, D., and Ke, H. (2005). Structure 13, this issue, 579–590. Louvion, J.F., Warth, R., and Picard, D. (1996). Proc. Natl. Acad. Sci. USA 93, 13937–13942. Meyer, P., Prodromou, C., Hu, B., Vaughan, C., Roe, S.M., Panaretou, B., Piper, P.W., and Pearl, L.H. (2003). Mol. Cell 11, 647–658. Prodromou, C., Roe, S.M., O’Brien, R., Ladbury, J.E., Piper, P.W., and Pearl, L.H. (1997). Cell 90, 65–75.
Selected Reading Ban, C., Junop, M., and Yang, W. (1999). Cell 97, 85–97. Brino, L., Urzhumtsev, A., Mousli, M., Bronner, C., Mitschler, A., Oudet, P., and Moras, D. (2000). J. Biol. Chem. 275, 9468–9475. Dutta, R., and Inouye, M. (2000). Trends Biochem. Sci. 25, 24–28. Harris, S.F., Shiau, A.K., and Agard, D.A. (2004). Structure 12, 1087–1097.
Stebbins, C.E., Russo, A.A., Schneider, C., Rosen, N., Hartl, F.U., and Pavletich, N.P. (1997). Cell 89, 239–250. Wegele, H., Müller, L., and Buchner, J. (2004). Rev. Physiol. Biochem. Pharmacol. 151, 1–44. Weikl, T., Muschler, P., Richter, K., Veit, T., Reinstein, J., and Buchner, J. (2000). J. Mol. Biol. 303, 583–592.
Structure, Vol. 13, April, 2005, ©2005 Elsevier Ltd All rights reserved. DOI 10.1016/j.str.2005.03.002
Unraveling the Mechanistic Details of Topoisomerases In this issue, Shuman and coworkers use methylphosphonates as a nonnative substrate to investigate the mechanism of type IB topoisomerases (Tian et al., 2005, this issue of Structure). A surprising gain-offunction result was critical to assigning a catalytic role to an active site arginine. DNA topoisomerases are important enzymes that alter the topology of DNA and are involved in many different DNA transactions. All topoisomerases perform a multistep catalytic cycle that involves cleavage of one or both DNA strands, passage of the strand(s) through the break, followed by religation of the cleaved DNA molecule. Topoisomerases form a transient phosphotyrosine intermediate with one end of the broken strand while the other end is kept sequestered in the molecule. Failure to complete the cycle would produce DNA damage that could result in cell death. This has lead to the development of a type of powerful chemotherapeutic that interferes with topoisomerases by stabilizing the DNA/ protein covalent intermediate and hence poisoning the cell (for reviews see Champoux, 2001; Corbett and Berger, 2004). Topoisomerases are classified into two types depending on whether they cleave one or both strands of the target DNA. Details of the mechanism of topoisomerases have emerged over the last few years via a variety of different experimental techniques, ranging from biochemical approaches to structural studies and, more recently, single-molecule studies (Corbett and Berger, 2004). Despite this, the cleavage and religation reactions are still not well understood for most topoisomerases due to the difficulty of studying a transient reaction in enzymes that display little or no sequence specificity. The exception is vaccinia virus topoisomerase I, a type IB topoisomerase. Type IB enzymes form a 3#-phosphotyrosyl intermediate and change the DNA
topology by a “controlled rotation” mechanism (Champoux, 2001). Structures of human and vaccinia virus topoisomerase I have shown that despite their great difference in size, the active site is essentially identical in both cases (Cheng et al., 1998; Redinbo et al., 1998). The structures of human topoisomerase I in covalent and noncovalent complex with DNA have helped pinpoint the nature of the protein/DNA interactions (Redinbo et al., 1998). The active site contains a catalytic pentad: the active site tyrosine, two arginines, a lysine, and a histidine (Corbett and Berger, 2004). Aside from the histidine, these residues are conserved in all type IB topoisomerases. Interestingly, type IB topoisomerases and tyrosine recombinases, such as flp and Cre, share a similar active site and most likely a cleavage/religation mechanism (Cheng et al., 1998). Vaccinia virus topoisomerase I has turned out to be an ideal system to study the details of the DNA transesterification reaction in type IB enzymes. Aside from being a small, easy to produce protein, it cleaves DNA at a sequence-specific site. More importantly, it is possible to use “suicide” substrates and other nonnative substrates that permit kinetic studies and detailed analysis of the effects of enzyme mutations. Recently, Shuman and collaborators introduced the use of modified oligonucleotides to study the details of the type IB topoisomerase cleavage reaction. In particular, the use of methylphosphonates, where the nonbridging oxygens in a phosphate group are replaced by methyl groups, has proven to be extremely powerful, as the charge of specific groups can be altered in predetermined ways without introducing a bulky chemical group. Using this approach, Shuman and coworkers showed that protein/DNA interactions at remote locations from the active site are important for active site assembly (Tian et al., 2004). Additionally, they observed that vaccinia virus topoisomerase I has a latent DNA nuclease activity that is revealed when the charge of the scissile phosphate is reduced (Tian et al., 2003). The effect is stereospecific: the Sp methylphosphonate has a much more dramatic effect than the Rp methylphosphonate on the hydrolysis reaction. The phenome-
Previews 503
non is explained by the electrostatic repulsion of water from the active site; once the repulsion is reduced, water can get in and allow hydrolysis of the tyrosinemethylphosphonate DNA bond. New results of Shuman and coworkers reported in this issue using methylphosphonates help further our understanding of the type IB topoisomerase transesterification reaction (Tian et al., 2005). Previously the role of one of the arginines in the catalytic pentad, R223 in vaccinia virus topoisomerase I, was undefined while the other arginine and a lysine were shown to be involved in expulsion of the 5#-oxygen of the leaving DNA strand. The importance of this arginine is underscored by the observation that replacement of the arginine with an alanine has a 105 effect on the transesterification reaction. Surprisingly, when the R223A mutant was studied using a Sp methylphosphonate at the scissile phosphate, a 200-fold increase in the cleavage of the modified substrate over the all-phosphodiester substrate was observed. Replacement of the arginine with a lysine recovered some of the transesterification activity compared to the alanine mutant. These two findings strongly support a role of the arginine in charge stabilization; the alanine mutant cannot contribute to charge buildup stabilization in the transition state, but the reduced charge of the methylphosphonate alleviates this need and allows for the reaction to proceed, albeit in a less efficient manner. Inserting an Rp methylphosphonate at the same position results in different kinetics that suggests an unexpected need for an Rp oxygen in the mutants, but not in the wild-type enzyme. Another surprising finding has to do with the religation reaction. Use of methylphosphonates showed that there is a modest requirement for a charge in the religation reaction, suggesting that ionic interactions are not as important for religation, probably because the proper alignment of the 5#-OH of the broken strand by protein/ DNA interactions is more important than any charge effect due to the phosphate. The results of Tian et al. (2005) help assign a role to a conserved arginine in the catalytic pentad: it is in-
volved in stabilization of the negative charge buildup in the phosphate group in the transition state. This result furthers our understanding of the catalytic mechanism of type IB topoisomerases and, by inference, of tyrosine recombinases. It also shows the importance of modifying the substrate, not only the enzyme, to address mechanistic questions. In this particular instance, the gain of function observed, namely the increase in hydrolysis of the phosphotyrosine intermediate, provided an invaluable observation needed to unravel the role of the arginine. Finally, it shows the power, and need, of a combined approach of structural biology and biochemistry to answer key questions in structure/ function relationships. The new knowledge garnered from the present work together with all the past work of many groups is starting to bring our understanding of topoisomerases to a level that only a few years ago was unthinkable. Alfonso Mondragón Department of Biochemistry, Molecular Biology, and Cell Biology Northwestern University 2205 Tech Drive Evanston, Illinois 60208
Selected Reading Champoux, J.J. (2001). Annu. Rev. Biochem. 70, 369–413. Cheng, C., Kussie, P., Pavletich, N., and Shuman, S. (1998). Cell 92, 841–850. Corbett, K.D., and Berger, J.M. (2004). Annu. Rev. Biophys. Biomol. Struct. 33, 95–118. Redinbo, M.R., Stewart, L., Kuhn, P., Champoux, J.J., and Hol, W.G. (1998). Science 279, 1504–1513. Tian, L., Claeboe, C.D., Hecht, S.M., and Shuman, S. (2003). Mol. Cell 12, 199–208. Tian, L., Claeboe, C.D., Hecht, S.M., and Shuman, S. (2004). Structure (Camb.) 12, 31–40. Tian, L., Claeboe, C.D., Hecht, S.M., and Shuman, S. (2005). Structure 13, this issue, 513–520.
Andreas Bracher and F. Ulrich Hartl Department of Cellular Biochemistry Max Planck Institute of Biochemistry Am Klopferspitz 18 82152 Martinsried Germany
Huai, Q., Wang, H., Liu, Y., Kim, H.-Y., Toft, D., and Ke, H. (2005). Structure 13, this issue, 579–590. Louvion, J.F., Warth, R., and Picard, D. (1996). Proc. Natl. Acad. Sci. USA 93, 13937–13942. Meyer, P., Prodromou, C., Hu, B., Vaughan, C., Roe, S.M., Panaretou, B., Piper, P.W., and Pearl, L.H. (2003). Mol. Cell 11, 647–658. Prodromou, C., Roe, S.M., O’Brien, R., Ladbury, J.E., Piper, P.W., and Pearl, L.H. (1997). Cell 90, 65–75.
Selected Reading Ban, C., Junop, M., and Yang, W. (1999). Cell 97, 85–97. Brino, L., Urzhumtsev, A., Mousli, M., Bronner, C., Mitschler, A., Oudet, P., and Moras, D. (2000). J. Biol. Chem. 275, 9468–9475. Dutta, R., and Inouye, M. (2000). Trends Biochem. Sci. 25, 24–28. Harris, S.F., Shiau, A.K., and Agard, D.A. (2004). Structure 12, 1087–1097.
Stebbins, C.E., Russo, A.A., Schneider, C., Rosen, N., Hartl, F.U., and Pavletich, N.P. (1997). Cell 89, 239–250. Wegele, H., Müller, L., and Buchner, J. (2004). Rev. Physiol. Biochem. Pharmacol. 151, 1–44. Weikl, T., Muschler, P., Richter, K., Veit, T., Reinstein, J., and Buchner, J. (2000). J. Mol. Biol. 303, 583–592.
Structure, Vol. 13, April, 2005, ©2005 Elsevier Ltd All rights reserved. DOI 10.1016/j.str.2005.03.002
Unraveling the Mechanistic Details of Topoisomerases In this issue, Shuman and coworkers use methylphosphonates as a nonnative substrate to investigate the mechanism of type IB topoisomerases (Tian et al., 2005, this issue of Structure). A surprising gain-offunction result was critical to assigning a catalytic role to an active site arginine. DNA topoisomerases are important enzymes that alter the topology of DNA and are involved in many different DNA transactions. All topoisomerases perform a multistep catalytic cycle that involves cleavage of one or both DNA strands, passage of the strand(s) through the break, followed by religation of the cleaved DNA molecule. Topoisomerases form a transient phosphotyrosine intermediate with one end of the broken strand while the other end is kept sequestered in the molecule. Failure to complete the cycle would produce DNA damage that could result in cell death. This has lead to the development of a type of powerful chemotherapeutic that interferes with topoisomerases by stabilizing the DNA/ protein covalent intermediate and hence poisoning the cell (for reviews see Champoux, 2001; Corbett and Berger, 2004). Topoisomerases are classified into two types depending on whether they cleave one or both strands of the target DNA. Details of the mechanism of topoisomerases have emerged over the last few years via a variety of different experimental techniques, ranging from biochemical approaches to structural studies and, more recently, single-molecule studies (Corbett and Berger, 2004). Despite this, the cleavage and religation reactions are still not well understood for most topoisomerases due to the difficulty of studying a transient reaction in enzymes that display little or no sequence specificity. The exception is vaccinia virus topoisomerase I, a type IB topoisomerase. Type IB enzymes form a 3#-phosphotyrosyl intermediate and change the DNA
topology by a “controlled rotation” mechanism (Champoux, 2001). Structures of human and vaccinia virus topoisomerase I have shown that despite their great difference in size, the active site is essentially identical in both cases (Cheng et al., 1998; Redinbo et al., 1998). The structures of human topoisomerase I in covalent and noncovalent complex with DNA have helped pinpoint the nature of the protein/DNA interactions (Redinbo et al., 1998). The active site contains a catalytic pentad: the active site tyrosine, two arginines, a lysine, and a histidine (Corbett and Berger, 2004). Aside from the histidine, these residues are conserved in all type IB topoisomerases. Interestingly, type IB topoisomerases and tyrosine recombinases, such as flp and Cre, share a similar active site and most likely a cleavage/religation mechanism (Cheng et al., 1998). Vaccinia virus topoisomerase I has turned out to be an ideal system to study the details of the DNA transesterification reaction in type IB enzymes. Aside from being a small, easy to produce protein, it cleaves DNA at a sequence-specific site. More importantly, it is possible to use “suicide” substrates and other nonnative substrates that permit kinetic studies and detailed analysis of the effects of enzyme mutations. Recently, Shuman and collaborators introduced the use of modified oligonucleotides to study the details of the type IB topoisomerase cleavage reaction. In particular, the use of methylphosphonates, where the nonbridging oxygens in a phosphate group are replaced by methyl groups, has proven to be extremely powerful, as the charge of specific groups can be altered in predetermined ways without introducing a bulky chemical group. Using this approach, Shuman and coworkers showed that protein/DNA interactions at remote locations from the active site are important for active site assembly (Tian et al., 2004). Additionally, they observed that vaccinia virus topoisomerase I has a latent DNA nuclease activity that is revealed when the charge of the scissile phosphate is reduced (Tian et al., 2003). The effect is stereospecific: the Sp methylphosphonate has a much more dramatic effect than the Rp methylphosphonate on the hydrolysis reaction. The phenome-
Previews 503
non is explained by the electrostatic repulsion of water from the active site; once the repulsion is reduced, water can get in and allow hydrolysis of the tyrosinemethylphosphonate DNA bond. New results of Shuman and coworkers reported in this issue using methylphosphonates help further our understanding of the type IB topoisomerase transesterification reaction (Tian et al., 2005). Previously the role of one of the arginines in the catalytic pentad, R223 in vaccinia virus topoisomerase I, was undefined while the other arginine and a lysine were shown to be involved in expulsion of the 5#-oxygen of the leaving DNA strand. The importance of this arginine is underscored by the observation that replacement of the arginine with an alanine has a 105 effect on the transesterification reaction. Surprisingly, when the R223A mutant was studied using a Sp methylphosphonate at the scissile phosphate, a 200-fold increase in the cleavage of the modified substrate over the all-phosphodiester substrate was observed. Replacement of the arginine with a lysine recovered some of the transesterification activity compared to the alanine mutant. These two findings strongly support a role of the arginine in charge stabilization; the alanine mutant cannot contribute to charge buildup stabilization in the transition state, but the reduced charge of the methylphosphonate alleviates this need and allows for the reaction to proceed, albeit in a less efficient manner. Inserting an Rp methylphosphonate at the same position results in different kinetics that suggests an unexpected need for an Rp oxygen in the mutants, but not in the wild-type enzyme. Another surprising finding has to do with the religation reaction. Use of methylphosphonates showed that there is a modest requirement for a charge in the religation reaction, suggesting that ionic interactions are not as important for religation, probably because the proper alignment of the 5#-OH of the broken strand by protein/ DNA interactions is more important than any charge effect due to the phosphate. The results of Tian et al. (2005) help assign a role to a conserved arginine in the catalytic pentad: it is in-
volved in stabilization of the negative charge buildup in the phosphate group in the transition state. This result furthers our understanding of the catalytic mechanism of type IB topoisomerases and, by inference, of tyrosine recombinases. It also shows the importance of modifying the substrate, not only the enzyme, to address mechanistic questions. In this particular instance, the gain of function observed, namely the increase in hydrolysis of the phosphotyrosine intermediate, provided an invaluable observation needed to unravel the role of the arginine. Finally, it shows the power, and need, of a combined approach of structural biology and biochemistry to answer key questions in structure/ function relationships. The new knowledge garnered from the present work together with all the past work of many groups is starting to bring our understanding of topoisomerases to a level that only a few years ago was unthinkable. Alfonso Mondragón Department of Biochemistry, Molecular Biology, and Cell Biology Northwestern University 2205 Tech Drive Evanston, Illinois 60208
Selected Reading Champoux, J.J. (2001). Annu. Rev. Biochem. 70, 369–413. Cheng, C., Kussie, P., Pavletich, N., and Shuman, S. (1998). Cell 92, 841–850. Corbett, K.D., and Berger, J.M. (2004). Annu. Rev. Biophys. Biomol. Struct. 33, 95–118. Redinbo, M.R., Stewart, L., Kuhn, P., Champoux, J.J., and Hol, W.G. (1998). Science 279, 1504–1513. Tian, L., Claeboe, C.D., Hecht, S.M., and Shuman, S. (2003). Mol. Cell 12, 199–208. Tian, L., Claeboe, C.D., Hecht, S.M., and Shuman, S. (2004). Structure (Camb.) 12, 31–40. Tian, L., Claeboe, C.D., Hecht, S.M., and Shuman, S. (2005). Structure 13, this issue, 513–520.