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. 1999 Feb 1;18(3):771–783. doi: 10.1093/emboj/18.3.771

The internal workings of a DNA polymerase clamp-loading machine.

J Turner 1, M M Hingorani 1, Z Kelman 1, M O'Donnell 1
PMCID: PMC1171170  PMID: 9927437

Abstract

Replicative DNA polymerases are multiprotein machines that are tethered to DNA during chain extension by sliding clamp proteins. The clamps are designed to encircle DNA completely, and they are manipulated rapidly onto DNA by the ATP-dependent activity of a clamp loader. We outline the detailed mechanism of gamma complex, a five-protein clamp loader that is part of the Escherichia coli replicase, DNA polymerase III holoenzyme. The gamma complex uses ATP to open the beta clamp and assemble it onto DNA. Surprisingly, ATP is not needed for gamma complex to crack open the beta clamp. The function of ATP is to regulate the activity of one subunit, delta, which opens the clamp simply by binding to it. The delta' subunit acts as a modulator of the interaction between delta and beta. On binding ATP, the gamma complex is activated such that the delta' subunit permits delta to bind beta and crack open the ring at one interface. The clamp loader-open clamp protein complex is now ready for an encounter with primed DNA to complete assembly of the clamp around DNA. Interaction with DNA stimulates ATP hydrolysis which ejects the gamma complex from DNA, leaving the ring to close around the duplex.

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Selected References

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  1. Blinkova A., Hervas C., Stukenberg P. T., Onrust R., O'Donnell M. E., Walker J. R. The Escherichia coli DNA polymerase III holoenzyme contains both products of the dnaX gene, tau and gamma, but only tau is essential. J Bacteriol. 1993 Sep;175(18):6018–6027. doi: 10.1128/jb.175.18.6018-6027.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blinkowa A. L., Walker J. R. Programmed ribosomal frameshifting generates the Escherichia coli DNA polymerase III gamma subunit from within the tau subunit reading frame. Nucleic Acids Res. 1990 Apr 11;18(7):1725–1729. doi: 10.1093/nar/18.7.1725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bloom L. B., Turner J., Kelman Z., Beechem J. M., O'Donnell M., Goodman M. F. Dynamics of loading the beta sliding clamp of DNA polymerase III onto DNA. J Biol Chem. 1996 Nov 29;271(48):30699–30708. doi: 10.1074/jbc.271.48.30699. [DOI] [PubMed] [Google Scholar]
  4. Burgers P. M., Kornberg A. ATP activation of DNA polymerase III holoenzyme from Escherichia coli. II. Initiation complex: stoichiometry and reactivity. J Biol Chem. 1982 Oct 10;257(19):11474–11478. [PubMed] [Google Scholar]
  5. Burgers P. M. Saccharomyces cerevisiae replication factor C. II. Formation and activity of complexes with the proliferating cell nuclear antigen and with DNA polymerases delta and epsilon. J Biol Chem. 1991 Nov 25;266(33):22698–22706. [PubMed] [Google Scholar]
  6. Burgers P. M., Yoder B. L. ATP-independent loading of the proliferating cell nuclear antigen requires DNA ends. J Biol Chem. 1993 Sep 25;268(27):19923–19926. [PubMed] [Google Scholar]
  7. Cai J., Gibbs E., Uhlmann F., Phillips B., Yao N., O'Donnell M., Hurwitz J. A complex consisting of human replication factor C p40, p37, and p36 subunits is a DNA-dependent ATPase and an intermediate in the assembly of the holoenzyme. J Biol Chem. 1997 Jul 25;272(30):18974–18981. doi: 10.1074/jbc.272.30.18974. [DOI] [PubMed] [Google Scholar]
  8. Cullmann G., Fien K., Kobayashi R., Stillman B. Characterization of the five replication factor C genes of Saccharomyces cerevisiae. Mol Cell Biol. 1995 Sep;15(9):4661–4671. doi: 10.1128/mcb.15.9.4661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dallmann H. G., McHenry C. S. DnaX complex of Escherichia coli DNA polymerase III holoenzyme. Physical characterization of the DnaX subunits and complexes. J Biol Chem. 1995 Dec 8;270(49):29563–29569. [PubMed] [Google Scholar]
  10. Dallmann H. G., Thimmig R. L., McHenry C. S. DnaX complex of Escherichia coli DNA polymerase III holoenzyme. Central role of tau in initiation complex assembly and in determining the functional asymmetry of holoenzyme. J Biol Chem. 1995 Dec 8;270(49):29555–29562. [PubMed] [Google Scholar]
  11. Dong Z., Onrust R., Skangalis M., O'Donnell M. DNA polymerase III accessory proteins. I. holA and holB encoding delta and delta'. J Biol Chem. 1993 Jun 5;268(16):11758–11765. [PubMed] [Google Scholar]
  12. Elledge S. J. Cell cycle checkpoints: preventing an identity crisis. Science. 1996 Dec 6;274(5293):1664–1672. doi: 10.1126/science.274.5293.1664. [DOI] [PubMed] [Google Scholar]
  13. Flower A. M., McHenry C. S. The gamma subunit of DNA polymerase III holoenzyme of Escherichia coli is produced by ribosomal frameshifting. Proc Natl Acad Sci U S A. 1990 May;87(10):3713–3717. doi: 10.1073/pnas.87.10.3713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Guenther B., Onrust R., Sali A., O'Donnell M., Kuriyan J. Crystal structure of the delta' subunit of the clamp-loader complex of E. coli DNA polymerase III. Cell. 1997 Oct 31;91(3):335–345. doi: 10.1016/s0092-8674(00)80417-1. [DOI] [PubMed] [Google Scholar]
  15. Gulbis J. M., Kelman Z., Hurwitz J., O'Donnell M., Kuriyan J. Structure of the C-terminal region of p21(WAF1/CIP1) complexed with human PCNA. Cell. 1996 Oct 18;87(2):297–306. doi: 10.1016/s0092-8674(00)81347-1. [DOI] [PubMed] [Google Scholar]
  16. Hingorani M. M., O'Donnell M. ATP binding to the Escherichia coli clamp loader powers opening of the ring-shaped clamp of DNA polymerase III holoenzyme. J Biol Chem. 1998 Sep 18;273(38):24550–24563. doi: 10.1074/jbc.273.38.24550. [DOI] [PubMed] [Google Scholar]
  17. Kelman Z., Naktinis V., O'Donnell M. Radiolabeling of proteins for biochemical studies. Methods Enzymol. 1995;262:430–442. doi: 10.1016/0076-6879(95)62034-6. [DOI] [PubMed] [Google Scholar]
  18. Kelman Z., O'Donnell M. DNA polymerase III holoenzyme: structure and function of a chromosomal replicating machine. Annu Rev Biochem. 1995;64:171–200. doi: 10.1146/annurev.bi.64.070195.001131. [DOI] [PubMed] [Google Scholar]
  19. Kelman Z., Yao N., O'Donnell M. Escherichia coli expression vectors containing a protein kinase recognition motif, His6-tag and hemagglutinin epitope. Gene. 1995 Dec 1;166(1):177–178. doi: 10.1016/0378-1119(95)00556-7. [DOI] [PubMed] [Google Scholar]
  20. Kelman Z., Yuzhakov A., Andjelkovic J., O'Donnell M. Devoted to the lagging strand-the subunit of DNA polymerase III holoenzyme contacts SSB to promote processive elongation and sliding clamp assembly. EMBO J. 1998 Apr 15;17(8):2436–2449. doi: 10.1093/emboj/17.8.2436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kim S., Dallmann H. G., McHenry C. S., Marians K. J. Coupling of a replicative polymerase and helicase: a tau-DnaB interaction mediates rapid replication fork movement. Cell. 1996 Feb 23;84(4):643–650. doi: 10.1016/s0092-8674(00)81039-9. [DOI] [PubMed] [Google Scholar]
  22. Kong X. P., Onrust R., O'Donnell M., Kuriyan J. Three-dimensional structure of the beta subunit of E. coli DNA polymerase III holoenzyme: a sliding DNA clamp. Cell. 1992 May 1;69(3):425–437. doi: 10.1016/0092-8674(92)90445-i. [DOI] [PubMed] [Google Scholar]
  23. Krishna T. S., Kong X. P., Gary S., Burgers P. M., Kuriyan J. Crystal structure of the eukaryotic DNA polymerase processivity factor PCNA. Cell. 1994 Dec 30;79(7):1233–1243. doi: 10.1016/0092-8674(94)90014-0. [DOI] [PubMed] [Google Scholar]
  24. Latham G. J., Pietroni P., Dong F., Young M. C., von Hippel P. H. Fluorescence monitoring of T4 polymerase holoenzyme accessory protein interactions during loading of the sliding clamp onto the template-primer junction. J Mol Biol. 1996 Dec 6;264(3):426–439. doi: 10.1006/jmbi.1996.0651. [DOI] [PubMed] [Google Scholar]
  25. Lee S. H., Hurwitz J. Mechanism of elongation of primed DNA by DNA polymerase delta, proliferating cell nuclear antigen, and activator 1. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5672–5676. doi: 10.1073/pnas.87.15.5672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Maki H., Maki S., Kornberg A. DNA Polymerase III holoenzyme of Escherichia coli. IV. The holoenzyme is an asymmetric dimer with twin active sites. J Biol Chem. 1988 May 15;263(14):6570–6578. [PubMed] [Google Scholar]
  27. Maki S., Kornberg A. DNA polymerase III holoenzyme of Escherichia coli. II. A novel complex including the gamma subunit essential for processive synthesis. J Biol Chem. 1988 May 15;263(14):6555–6560. [PubMed] [Google Scholar]
  28. McHenry C. S. Purification and characterization of DNA polymerase III'. Identification of tau as a subunit of the DNA polymerase III holoenzyme. J Biol Chem. 1982 Mar 10;257(5):2657–2663. [PubMed] [Google Scholar]
  29. Mossi R., Hübscher U. Clamping down on clamps and clamp loaders--the eukaryotic replication factor C. Eur J Biochem. 1998 Jun 1;254(2):209–216. [PubMed] [Google Scholar]
  30. Naktinis V., Onrust R., Fang L., O'Donnell M. Assembly of a chromosomal replication machine: two DNA polymerases, a clamp loader, and sliding clamps in one holoenzyme particle. II. Intermediate complex between the clamp loader and its clamp. J Biol Chem. 1995 Jun 2;270(22):13358–13365. [PubMed] [Google Scholar]
  31. Naktinis V., Turner J., O'Donnell M. A molecular switch in a replication machine defined by an internal competition for protein rings. Cell. 1996 Jan 12;84(1):137–145. doi: 10.1016/s0092-8674(00)81000-4. [DOI] [PubMed] [Google Scholar]
  32. Noskov V. N., Araki H., Sugino A. The RFC2 gene, encoding the third-largest subunit of the replication factor C complex, is required for an S-phase checkpoint in Saccharomyces cerevisiae. Mol Cell Biol. 1998 Aug;18(8):4914–4923. doi: 10.1128/mcb.18.8.4914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. O'Donnell M. E. Accessory proteins bind a primed template and mediate rapid cycling of DNA polymerase III holoenzyme from Escherichia coli. J Biol Chem. 1987 Dec 5;262(34):16558–16565. [PubMed] [Google Scholar]
  34. Onrust R., Finkelstein J., Naktinis V., Turner J., Fang L., O'Donnell M. Assembly of a chromosomal replication machine: two DNA polymerases, a clamp loader, and sliding clamps in one holoenzyme particle. I. Organization of the clamp loader. J Biol Chem. 1995 Jun 2;270(22):13348–13357. doi: 10.1074/jbc.270.22.13348. [DOI] [PubMed] [Google Scholar]
  35. Onrust R., Finkelstein J., Turner J., Naktinis V., O'Donnell M. Assembly of a chromosomal replication machine: two DNA polymerases, a clamp loader, and sliding clamps in one holoenzyme particle. III. Interface between two polymerases and the clamp loader. J Biol Chem. 1995 Jun 2;270(22):13366–13377. doi: 10.1074/jbc.270.22.13366. [DOI] [PubMed] [Google Scholar]
  36. Onrust R., O'Donnell M. DNA polymerase III accessory proteins. II. Characterization of delta and delta'. J Biol Chem. 1993 Jun 5;268(16):11766–11772. [PubMed] [Google Scholar]
  37. Onrust R., Stukenberg P. T., O'Donnell M. Analysis of the ATPase subassembly which initiates processive DNA synthesis by DNA polymerase III holoenzyme. J Biol Chem. 1991 Nov 15;266(32):21681–21686. [PubMed] [Google Scholar]
  38. Perkins G., Diffley J. F. Nucleotide-dependent prereplicative complex assembly by Cdc6p, a homolog of eukaryotic and prokaryotic clamp-loaders. Mol Cell. 1998 Jul;2(1):23–32. doi: 10.1016/s1097-2765(00)80110-0. [DOI] [PubMed] [Google Scholar]
  39. Pietroni P., Young M. C., Latham G. J., von Hippel P. H. Structural analyses of gp45 sliding clamp interactions during assembly of the bacteriophage T4 DNA polymerase holoenzyme. I. Conformational changes within the gp44/62-gp45-ATP complex during clamp loading. J Biol Chem. 1997 Dec 12;272(50):31666–31676. doi: 10.1074/jbc.272.50.31666. [DOI] [PubMed] [Google Scholar]
  40. Sexton D. J., Carver T. E., Berdis A. J., Benkovic S. J. Protein-protein and protein-DNA interactions at the bacteriophage T4 DNA replication fork. Characterization of a fluorescently labeled DNA polymerase sliding clamp. J Biol Chem. 1996 Nov 8;271(45):28045–28051. doi: 10.1074/jbc.271.45.28045. [DOI] [PubMed] [Google Scholar]
  41. Studwell-Vaughan P. S., O'Donnell M. Constitution of the twin polymerase of DNA polymerase III holoenzyme. J Biol Chem. 1991 Oct 15;266(29):19833–19841. [PubMed] [Google Scholar]
  42. Studwell P. S., O'Donnell M. Processive replication is contingent on the exonuclease subunit of DNA polymerase III holoenzyme. J Biol Chem. 1990 Jan 15;265(2):1171–1178. [PubMed] [Google Scholar]
  43. Stukenberg P. T., Studwell-Vaughan P. S., O'Donnell M. Mechanism of the sliding beta-clamp of DNA polymerase III holoenzyme. J Biol Chem. 1991 Jun 15;266(17):11328–11334. [PubMed] [Google Scholar]
  44. Stukenberg P. T., Turner J., O'Donnell M. An explanation for lagging strand replication: polymerase hopping among DNA sliding clamps. Cell. 1994 Sep 9;78(5):877–887. doi: 10.1016/s0092-8674(94)90662-9. [DOI] [PubMed] [Google Scholar]
  45. Sugimoto K., Shimomura T., Hashimoto K., Araki H., Sugino A., Matsumoto K. Rfc5, a small subunit of replication factor C complex, couples DNA replication and mitosis in budding yeast. Proc Natl Acad Sci U S A. 1996 Jul 9;93(14):7048–7052. doi: 10.1073/pnas.93.14.7048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Tsuchihashi Z., Kornberg A. ATP interactions of the tau and gamma subunits of DNA polymerase III holoenzyme of Escherichia coli. J Biol Chem. 1989 Oct 25;264(30):17790–17795. [PubMed] [Google Scholar]
  47. Turner J., O'Donnell M. Cycling of Escherichia coli DNA polymerase III from one sliding clamp to another: model for lagging strand. Methods Enzymol. 1995;262:442–449. doi: 10.1016/0076-6879(95)62035-4. [DOI] [PubMed] [Google Scholar]
  48. Wickner S. Mechanism of DNA elongation catalyzed by Escherichia coli DNA polymerase III, dnaZ protein, and DNA elongation factors I and III. Proc Natl Acad Sci U S A. 1976 Oct;73(10):3511–3515. doi: 10.1073/pnas.73.10.3511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Xiao H., Dong Z., O'Donnell M. DNA polymerase III accessory proteins. IV. Characterization of chi and psi. J Biol Chem. 1993 Jun 5;268(16):11779–11784. [PubMed] [Google Scholar]
  50. Xiao H., Naktinis V., O'Donnell M. Assembly of a chromosomal replication machine: two DNA polymerases, a clamp loader, and sliding clamps in one holoenzyme particle. IV. ATP-binding site mutants identify the clamp loader. J Biol Chem. 1995 Jun 2;270(22):13378–13383. doi: 10.1074/jbc.270.22.13378. [DOI] [PubMed] [Google Scholar]
  51. Yao N., Turner J., Kelman Z., Stukenberg P. T., Dean F., Shechter D., Pan Z. Q., Hurwitz J., O'Donnell M. Clamp loading, unloading and intrinsic stability of the PCNA, beta and gp45 sliding clamps of human, E. coli and T4 replicases. Genes Cells. 1996 Jan;1(1):101–113. doi: 10.1046/j.1365-2443.1996.07007.x. [DOI] [PubMed] [Google Scholar]
  52. Yuzhakov A., Kelman Z., O'Donnell M. Trading places on DNA--a three-point switch underlies primer handoff from primase to the replicative DNA polymerase. Cell. 1999 Jan 8;96(1):153–163. doi: 10.1016/s0092-8674(00)80968-x. [DOI] [PubMed] [Google Scholar]
  53. Yuzhakov A., Turner J., O'Donnell M. Replisome assembly reveals the basis for asymmetric function in leading and lagging strand replication. Cell. 1996 Sep 20;86(6):877–886. doi: 10.1016/s0092-8674(00)80163-4. [DOI] [PubMed] [Google Scholar]

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