Cell Cycle
ISSN: 1538-4101 (Print) 1551-4005 (Online) Journal homepage: http://www.tandfonline.com/loi/kccy20
Watching a DNA polymerase in action Bret D Freudenthal, William A Beard & Samuel H Wilson To cite this article: Bret D Freudenthal, William A Beard & Samuel H Wilson (2014) Watching a DNA polymerase in action, Cell Cycle, 13:5, 691-692, DOI: 10.4161/cc.27789 To link to this article: http://dx.doi.org/10.4161/cc.27789
Copyright © 2014 Landes Bioscience
Published online: 14 Jan 2014.
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Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=kccy20 Download by: [111.237.162.108]
Date: 12 September 2015, At: 23:02
Editorials: Cell Cycle Features
Editorials: Cell Cycle Features
Cell Cycle 13:5, 691–692; March 1, 2014; © 2014 Landes Bioscience
Watching a DNA polymerase in action Bret D Freudenthal, William A Beard, and Samuel H Wilson*
Downloaded by [111.237.162.108] at 23:02 12 September 2015
Laboratory of Structural Biology; National Institute of Environmental Health Sciences; NIH; Research Triangle Park, NC USA
DNA polymerase (pol) β is a model eukaryotic DNA polymerase due to its small size, stability in structural studies, and significant biological role during DNA repair. Previous studies had determined that pol β binds to single-nucleotide gapped DNA in an open conformation. Upon binding an incoming nucleoside triphosphate and 2 metal ions, pol β undergoes a conformational change to form a pre-catalytic closed complex. If a nucleotide binds that preserves Watson–Crick hydrogen bonding, the complex is optimized for DNA synthesis, with 2 metal ions facilitating chemistry. Following catalysis, pol β relaxes to an open conformation, releasing PPi and the extended DNA. This mechanism is consistent with results from structural, computational, biochemical, and kinetic experiments.1 However, to characterize key intermediate states during DNA synthesis, structural snapshots of these intermediates are required. Emerging structural techniques have provided a means to capture these snapshots, allowing new opportunities for dissecting the strategies polymerases use in protecting the genome.2,3 Photographers have utilized time-lapse snapshots to capture singular events and place them in the context of larger experiences. In a similar way, we have captured intermediate snapshots of the DNA polymerase reaction, utilizing the time-lapse or time-resolved crystallography technique with pol β3. This approach uses natural substrates to overcome limitations of artificially trapping intermediates with abortive nucleotide analogs.4 We first form the closed pre-insertion substrate complex of pol β, DNA, and the incoming nucleotide in the presence of Ca 2+. Calcium prevents catalysis, while allowing substrates to bind in the normal fashion. We then initiate
the reaction by transferring these crystals into a solution containing MgCl2 for various time intervals and stop the reaction by flash freezing. The crystals provide for high-resolution (1.8–2 Å) structural determinations representing snapshots along the reaction pathway. Thus, depending on the soak time prior to data collection, we can observe steps before, during, and after nucleotide insertion at the atomic level. Using the time-lapse crystallography approach with right and wrong nucleotides allowed us to capture key steps during catalysis, providing molecular insights by which polymerases optimize fidelity.3 Importantly, we observed key conformational adjustments in the polymerase and substrates that hasten correct and deter incorrect insertion. This is consistent with the induced fit model, in which conformational changes optimize the active site for correct nucleotide insertion, but distort the active site after incorrect nucleotide binding.5 For example, conformational closing induced by incorrect nucleotide binding re-orients the primer strand to a less favorable catalytic position and distorts the catalytic metal binding pocket. Long incubations indicated that the pyrophosphate (PPi) product was released more rapidly following incorrect, compared with correct, nucleotide insertion. This product release is coupled to subdomain repositioning (closed to open) that may impact downstream repair events in large, multi-protein DNA repair complexes. These multi-protein complexes facilitate the channeling (i.e., direct transfer) of toxic DNA intermediates between repair proteins. This channeling has been shown to occur between apurinic/apyrimidinic endonuclease 1 (Ape1), pol β, and DNA ligase 1 during the repair of simple DNA lesions.6 The conformational
dichotomy observed after insertion of right or wrong nucleotides suggest that the conformation of pol β (open vs. closed states) could couple the channeling of toxic DNA intermediates. In this model, insertion of the correct nucleotide by pol β into a single-nucleotide gap channels the nicked DNA product to DNA ligase. This would be encouraged by the closed polymerase complex immediately after nucleotide insertion. In contrast, following incorrect insertion, pol β rapidly reopens, channeling the substrate to Ape1 for correction of the mismatch utilizing the proofreading activity of Ape1.7 In this scenario, the open and closed state of an enzyme in a multi-protein repair complex provides a structural signal requesting the appropriate enzyme for successful substrate channeling and DNA repair. This is an exciting, yet untested idea that will have implications for controlling DNA damage processing and pathway differentiation at the molecular level. DNA polymerases use a 2-metal catalytic mechanism during DNA synthesis.8 Recently, a third metal ion was observed in the active site of a DNA polymerase involved in DNA damage bypass.2 Using the time-lapse crystallography technique, we also observed a third metal ion binding site following catalysis of the correct, but not incorrect nucleotide.3 This transient metal-binding site in the product complex was only observed with bound PPi, and we proposed it being involved in facilitating the reverse reaction, or pyrophospholysis. Accordingly, a 2-metal ion mechanism for the forward and reverse reaction is proposed: both reactions utilize a nucleotide or PPi metal-binding site, while the forward reaction uses the traditional catalytic metal-binding site, and the reverse reaction uses the third metal-binding site.
*Correspondence to: Samuel H Wilson; Email: [email protected] Submitted: 10/30/2013; Accepted: 11/04/2013 http://dx.doi.org/10.4161/cc.27789 Comment on: Freudenthal BD, et al. Cell 2013; 154:157-68; PMID:23827680; http://dx.doi.org/10.1016/j.cell.2013.05.048
www.landesbioscience.com
Cell Cycle 691
dynamic events that can be biologically important. References 1. Beard WA, et al. Chem Rev 2006; 106:361-82; PMID:16464010; http://dx.doi.org/10.1021/ cr0404904 2. Nakamura T, et al. Nature 2012; 487:196-201; PMID:22785315; http://dx.doi.org/10.1038/ nature11181 3. Freudenthal BD, et al. Cell 2013; 154:157-68; PMID:23827680; http://dx.doi.org/10.1016/j. cell.2013.05.048
4. Batra VK, et al. Structure 2006; 14:757-66; PMID:16615916; http://dx.doi.org/10.1016/j. str.2006.01.011 5. Johnson KA. J Biol Chem 2008; 283:26297-301; PMID:18544537; http://dx.doi.org/10.1074/jbc. R800034200 6. Prasad R, et al. J Biol Chem 2010; 285:40479-88; PMID:20952393; http://dx.doi.org/10.1074/jbc. M110.155267 7. Chou K-M, et al. Nature 2002; 415:6559; PMID:11832948; http://dx.doi. org/10.1038/415655a 8. Beese LS, et al. EMBO J 1991; 10:25-33; PMID:1989886
Downloaded by [111.237.162.108] at 23:02 12 September 2015
Subsequent studies will be required to probe the role of this novel third metalbinding site. Developing time-lapse crystallography with pol β using natural substrates provides an approach that can be applied to many enzyme systems. Capturing these intermediate snapshots highlights the dynamic nature of ligand binding and catalysis and provides a new structural tool that will help bridge the static snapshots of X-ray crystallography with the
692
Cell Cycle
Volume 13 Issue 5
ISSN: 1538-4101 (Print) 1551-4005 (Online) Journal homepage: http://www.tandfonline.com/loi/kccy20
Watching a DNA polymerase in action Bret D Freudenthal, William A Beard & Samuel H Wilson To cite this article: Bret D Freudenthal, William A Beard & Samuel H Wilson (2014) Watching a DNA polymerase in action, Cell Cycle, 13:5, 691-692, DOI: 10.4161/cc.27789 To link to this article: http://dx.doi.org/10.4161/cc.27789
Copyright © 2014 Landes Bioscience
Published online: 14 Jan 2014.
Submit your article to this journal
Article views: 48
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=kccy20 Download by: [111.237.162.108]
Date: 12 September 2015, At: 23:02
Editorials: Cell Cycle Features
Editorials: Cell Cycle Features
Cell Cycle 13:5, 691–692; March 1, 2014; © 2014 Landes Bioscience
Watching a DNA polymerase in action Bret D Freudenthal, William A Beard, and Samuel H Wilson*
Downloaded by [111.237.162.108] at 23:02 12 September 2015
Laboratory of Structural Biology; National Institute of Environmental Health Sciences; NIH; Research Triangle Park, NC USA
DNA polymerase (pol) β is a model eukaryotic DNA polymerase due to its small size, stability in structural studies, and significant biological role during DNA repair. Previous studies had determined that pol β binds to single-nucleotide gapped DNA in an open conformation. Upon binding an incoming nucleoside triphosphate and 2 metal ions, pol β undergoes a conformational change to form a pre-catalytic closed complex. If a nucleotide binds that preserves Watson–Crick hydrogen bonding, the complex is optimized for DNA synthesis, with 2 metal ions facilitating chemistry. Following catalysis, pol β relaxes to an open conformation, releasing PPi and the extended DNA. This mechanism is consistent with results from structural, computational, biochemical, and kinetic experiments.1 However, to characterize key intermediate states during DNA synthesis, structural snapshots of these intermediates are required. Emerging structural techniques have provided a means to capture these snapshots, allowing new opportunities for dissecting the strategies polymerases use in protecting the genome.2,3 Photographers have utilized time-lapse snapshots to capture singular events and place them in the context of larger experiences. In a similar way, we have captured intermediate snapshots of the DNA polymerase reaction, utilizing the time-lapse or time-resolved crystallography technique with pol β3. This approach uses natural substrates to overcome limitations of artificially trapping intermediates with abortive nucleotide analogs.4 We first form the closed pre-insertion substrate complex of pol β, DNA, and the incoming nucleotide in the presence of Ca 2+. Calcium prevents catalysis, while allowing substrates to bind in the normal fashion. We then initiate
the reaction by transferring these crystals into a solution containing MgCl2 for various time intervals and stop the reaction by flash freezing. The crystals provide for high-resolution (1.8–2 Å) structural determinations representing snapshots along the reaction pathway. Thus, depending on the soak time prior to data collection, we can observe steps before, during, and after nucleotide insertion at the atomic level. Using the time-lapse crystallography approach with right and wrong nucleotides allowed us to capture key steps during catalysis, providing molecular insights by which polymerases optimize fidelity.3 Importantly, we observed key conformational adjustments in the polymerase and substrates that hasten correct and deter incorrect insertion. This is consistent with the induced fit model, in which conformational changes optimize the active site for correct nucleotide insertion, but distort the active site after incorrect nucleotide binding.5 For example, conformational closing induced by incorrect nucleotide binding re-orients the primer strand to a less favorable catalytic position and distorts the catalytic metal binding pocket. Long incubations indicated that the pyrophosphate (PPi) product was released more rapidly following incorrect, compared with correct, nucleotide insertion. This product release is coupled to subdomain repositioning (closed to open) that may impact downstream repair events in large, multi-protein DNA repair complexes. These multi-protein complexes facilitate the channeling (i.e., direct transfer) of toxic DNA intermediates between repair proteins. This channeling has been shown to occur between apurinic/apyrimidinic endonuclease 1 (Ape1), pol β, and DNA ligase 1 during the repair of simple DNA lesions.6 The conformational
dichotomy observed after insertion of right or wrong nucleotides suggest that the conformation of pol β (open vs. closed states) could couple the channeling of toxic DNA intermediates. In this model, insertion of the correct nucleotide by pol β into a single-nucleotide gap channels the nicked DNA product to DNA ligase. This would be encouraged by the closed polymerase complex immediately after nucleotide insertion. In contrast, following incorrect insertion, pol β rapidly reopens, channeling the substrate to Ape1 for correction of the mismatch utilizing the proofreading activity of Ape1.7 In this scenario, the open and closed state of an enzyme in a multi-protein repair complex provides a structural signal requesting the appropriate enzyme for successful substrate channeling and DNA repair. This is an exciting, yet untested idea that will have implications for controlling DNA damage processing and pathway differentiation at the molecular level. DNA polymerases use a 2-metal catalytic mechanism during DNA synthesis.8 Recently, a third metal ion was observed in the active site of a DNA polymerase involved in DNA damage bypass.2 Using the time-lapse crystallography technique, we also observed a third metal ion binding site following catalysis of the correct, but not incorrect nucleotide.3 This transient metal-binding site in the product complex was only observed with bound PPi, and we proposed it being involved in facilitating the reverse reaction, or pyrophospholysis. Accordingly, a 2-metal ion mechanism for the forward and reverse reaction is proposed: both reactions utilize a nucleotide or PPi metal-binding site, while the forward reaction uses the traditional catalytic metal-binding site, and the reverse reaction uses the third metal-binding site.
*Correspondence to: Samuel H Wilson; Email: [email protected] Submitted: 10/30/2013; Accepted: 11/04/2013 http://dx.doi.org/10.4161/cc.27789 Comment on: Freudenthal BD, et al. Cell 2013; 154:157-68; PMID:23827680; http://dx.doi.org/10.1016/j.cell.2013.05.048
www.landesbioscience.com
Cell Cycle 691
dynamic events that can be biologically important. References 1. Beard WA, et al. Chem Rev 2006; 106:361-82; PMID:16464010; http://dx.doi.org/10.1021/ cr0404904 2. Nakamura T, et al. Nature 2012; 487:196-201; PMID:22785315; http://dx.doi.org/10.1038/ nature11181 3. Freudenthal BD, et al. Cell 2013; 154:157-68; PMID:23827680; http://dx.doi.org/10.1016/j. cell.2013.05.048
4. Batra VK, et al. Structure 2006; 14:757-66; PMID:16615916; http://dx.doi.org/10.1016/j. str.2006.01.011 5. Johnson KA. J Biol Chem 2008; 283:26297-301; PMID:18544537; http://dx.doi.org/10.1074/jbc. R800034200 6. Prasad R, et al. J Biol Chem 2010; 285:40479-88; PMID:20952393; http://dx.doi.org/10.1074/jbc. M110.155267 7. Chou K-M, et al. Nature 2002; 415:6559; PMID:11832948; http://dx.doi. org/10.1038/415655a 8. Beese LS, et al. EMBO J 1991; 10:25-33; PMID:1989886
Downloaded by [111.237.162.108] at 23:02 12 September 2015
Subsequent studies will be required to probe the role of this novel third metalbinding site. Developing time-lapse crystallography with pol β using natural substrates provides an approach that can be applied to many enzyme systems. Capturing these intermediate snapshots highlights the dynamic nature of ligand binding and catalysis and provides a new structural tool that will help bridge the static snapshots of X-ray crystallography with the
692
Cell Cycle
Volume 13 Issue 5
