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. 1980 Jun;77(6):3312–3316. doi: 10.1073/pnas.77.6.3312

Multienzyme complex for metabolic channeling in mammalian DNA replication.

G Prem veer Reddy, A B Pardee
PMCID: PMC349605  PMID: 6251456

Abstract

In the DNA-synthesizing phase (S phase) of CHEF/18 Chinese hamster embryo fibroblast cells, six enzymes associated with DNA metabolism, including DNA polymerase (deoxynucleoside triphosphate:DNA deoxynucleotidyl-transferase, EC 2.7.7.7), were largely localized in the nuclear region (karyoplasts). By contrast, in quiescent and G1 phase cells these enzymatic activites were mainly absent from the nucleus and were recovered in the cytoplasmic portion (cytoplasts). These nuclear (but not cytoplasmic) enzymatic activities cosedimented rapidly on sucrose density gradients. Further, the rapidly sedimenting enzyme activities were unique to cells in S phase. An organized supramolecular structure that allows channeling of metabolites into DNA was demonstrated by kinetics of nucleotide incorporation. "Permeabilized" cells selectively channeled incorporation of ribonucleoside diphosphates into DNA in preference to deoxyribonucleoside triphosphates. Deoxyribonucleoside triphosphate incorporation occurred when ribonucleoside-diphosphate reductase (2'-deoxyribonucleoside-diphosphate: oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1) activity was abolished by hydroxyurea. Our interpretation is that during DNA replication, the nucleus contains a complex of DNA precursor-synthesizing enzymes juxtaposed with the "replication apparatus" comprising DNA polymerase, other enzymes, and structural proteins. Functional integrity of this structure is impaired when one of its essential components is inactivated. We propose the name "replitase" for this multienzyme complex for DNA replication and suggest that it incorporates precursors rapidly and efficiently. Possibly its assembly signals the initiation of the S phase of the cell cycle.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bogenhagen D., Clayton D. A. Thymidylate nucleotide supply for mitochondrial DNA synthesis in mouse L-cells. Effect of 5-fluorodeoxyuridine and methotrexate in thymidine kinase plus and thymidine kinase minus cells. J Biol Chem. 1976 May 25;251(10):2938–2944. [PubMed] [Google Scholar]
  2. Castellot J. J., Jr, Miller M. R., Lehtomaki D. M., Pardee A. B. Comparison of DNA replication and repair enzymology using permeabilized baby hamster kidney cells. J Biol Chem. 1979 Aug 10;254(15):6904–6908. [PubMed] [Google Scholar]
  3. Castellot J. J., Jr, Miller M. R., Pardee A. B. Animal cells reversibly permeable to small molecules. Proc Natl Acad Sci U S A. 1978 Jan;75(1):351–355. doi: 10.1073/pnas.75.1.351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cherington P. V., Smith B. L., Pardee A. B. Loss of epidermal growth factor requirement and malignant transformation. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3937–3941. doi: 10.1073/pnas.76.8.3937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Collins J. M. Rates of DNA synthesis during the S-phase of HeLa cells. J Biol Chem. 1978 Dec 10;253(23):8570–8577. [PubMed] [Google Scholar]
  6. Comings D. E., Kakefuda T. Initiation of deoxyribonucleic acid replication at the nuclear membrane in human cells. J Mol Biol. 1968 Apr 14;33(1):225–229. doi: 10.1016/0022-2836(68)90290-8. [DOI] [PubMed] [Google Scholar]
  7. Duckworth D. H., Bessman M. J. The enzymology of virus-infected bacteria. X. A biochemical-genetic study of the deoxynucleotide kinase induced by wild type and amber mutants of phage T4. J Biol Chem. 1967 Jun 25;242(12):2877–2885. [PubMed] [Google Scholar]
  8. Elford H. L., Freese M., Passamani E., Morris H. P. Ribonucleotide reductase and cell proliferation. I. Variations of ribonucleotide reductase activity with tumor growth rate in a series of rat hepatomas. J Biol Chem. 1970 Oct 25;245(20):5228–5233. [PubMed] [Google Scholar]
  9. Engström Y., Eriksson S., Thelander L., Akerman M. Ribonucleotide reductase from calf thymus. Purification and properties. Biochemistry. 1979 Jul 10;18(14):2941–2948. doi: 10.1021/bi00581a004. [DOI] [PubMed] [Google Scholar]
  10. Firshein W. In situ activity of enzymes on polyacrylamide gels of a deoxyribonucleic acid-membrane fraction extracted from pneumococci. J Bacteriol. 1974 Jun;118(3):1101–1110. doi: 10.1128/jb.118.3.1101-1110.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Flanegan J. B., Greenberg G. R. Regulation of deoxyribonucleotide biosynthesis during in vivo bacteriophage T4 DNA replication. Intrinsic control of synthesis of thymine and 5-hydroxymethylcytosine deoxyribonucleotides at precise ratio found in DNA. J Biol Chem. 1977 May 10;252(9):3019–3027. [PubMed] [Google Scholar]
  12. Fridland A. DNA precursors in eukaryotic cells. Nat New Biol. 1973 May 23;243(125):105–107. [PubMed] [Google Scholar]
  13. Greene M., Firshein W. Role of deoxyribonucleic acid ligase in a doxyribonucleic acid membrane fraction extracted from pneumococci. J Bacteriol. 1976 May;126(2):777–784. doi: 10.1128/jb.126.2.777-784.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hamlin J. L., Pardee A. B. S phase synchrony in monolayer CHO cultures. Exp Cell Res. 1976 Jul;100(2):265–275. doi: 10.1016/0014-4827(76)90147-6. [DOI] [PubMed] [Google Scholar]
  15. Herrick G., Spear B. B., Veomett G. Intracellular localization of mouse DNA polymerase-alpha. Proc Natl Acad Sci U S A. 1976 Apr;73(4):1136–1139. doi: 10.1073/pnas.73.4.1136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Infante A. A., Firshein W., Hobart P., Murray L. A nuclear membrane-associated DNA complex in cultured mammalian cells capable of synthesizing DNA in vitro. Biochemistry. 1976 Nov 2;15(22):4810–4817. doi: 10.1021/bi00667a010. [DOI] [PubMed] [Google Scholar]
  17. Infante A. A., Nauta R., Gilbert S., Hobart P., Firshein W. DNA synthesis in developing sea urchins: role of a DNA-nuclear membrane complex. Nat New Biol. 1973 Mar 7;242(114):5–8. doi: 10.1038/newbio242005a0. [DOI] [PubMed] [Google Scholar]
  18. Kornberg T., Gefter M. L. Deoxyribonucleic acid synthesis in cell-free extracts. IV. Purification and catalytic properties of deoxyribonucleic acid polymerase III. J Biol Chem. 1972 Sep 10;247(17):5369–5375. [PubMed] [Google Scholar]
  19. Krokan H., Bjorklid E., Prydz H. DNA synthesis in isolated HeLa cell nuclei. Optimalization of the system and characterization of the product. Biochemistry. 1975 Sep 23;14(19):4227–4232. doi: 10.1021/bi00690a012. [DOI] [PubMed] [Google Scholar]
  20. Kuebbing D., Werner R. A model for compartmentation of de novo and salvage thymidine nucleotide pools in mammalian cells. Proc Natl Acad Sci U S A. 1975 Sep;72(9):3333–3336. doi: 10.1073/pnas.72.9.3333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  22. Larsson A. Ribonucleotide reductase rom regenerating rat liver. Eur J Biochem. 1969 Nov;11(1):113–121. doi: 10.1111/j.1432-1033.1969.tb00747.x. [DOI] [PubMed] [Google Scholar]
  23. LeBlanc D. J., Singer M. F. Localization of replicating DNA of simian virus 40 in monkey kidney cells. Proc Natl Acad Sci U S A. 1974 Jun;71(6):2236–2240. doi: 10.1073/pnas.71.6.2236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lewis W. H., Kuzik B. A., Wright J. A. Assay of ribonucleotide reduction in nucleotide-permeable hamster cells. J Cell Physiol. 1978 Mar;94(3):287–298. doi: 10.1002/jcp.1040940306. [DOI] [PubMed] [Google Scholar]
  25. Lockshin A., Moran R. G., Danenberg P. V. Thymidylate synthetase purified to homogeneity from human leukemic cells. Proc Natl Acad Sci U S A. 1979 Feb;76(2):750–754. doi: 10.1073/pnas.76.2.750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lucas J. J., Kates J. R. The construction of viable nuclear-cytoplasmic hybrid cells by nuclear transplantation. Cell. 1976 Mar;7(3):397–405. doi: 10.1016/0092-8674(76)90169-0. [DOI] [PubMed] [Google Scholar]
  27. Miller M. R., Castellot J. J., Jr, Pardee A. B. A permeable animal cell preparation for studying macromolecular synthesis. DNA synthesis and the role of deoxyribonucleotides in S phase initiation. Biochemistry. 1978 Mar 21;17(6):1073–1080. doi: 10.1021/bi00599a021. [DOI] [PubMed] [Google Scholar]
  28. Mizuno N. S., Stoops C. E., Sinha A. A. DNA synthesis associated with the inner membrane of the nuclear envelope. Nat New Biol. 1971 Jan 6;229(1):22–24. doi: 10.1038/newbio229022a0. [DOI] [PubMed] [Google Scholar]
  29. Mosbach K., Mattiasson B. Immobilized model systems of enzyme sequences. Curr Top Cell Regul. 1978;14:197–241. doi: 10.1016/b978-0-12-152814-0.50009-5. [DOI] [PubMed] [Google Scholar]
  30. Nickerson J. A., Wells W. W. Association of nucleosidediphosphate kinase with microtubules. Biochem Biophys Res Commun. 1978 Nov 29;85(2):820–826. doi: 10.1016/0006-291x(78)91236-6. [DOI] [PubMed] [Google Scholar]
  31. O'Brien R. L., Sanyal A. B., Stanton R. H. Association of DNA replication with the nuclear membrane of HeLa cells. Exp Cell Res. 1972 Jan;70(1):106–112. doi: 10.1016/0014-4827(72)90187-5. [DOI] [PubMed] [Google Scholar]
  32. Pardoll D. M., Vogelstein B., Coffey D. S. A fixed site of DNA replication in eucaryotic cells. Cell. 1980 Feb;19(2):527–536. doi: 10.1016/0092-8674(80)90527-9. [DOI] [PubMed] [Google Scholar]
  33. Pato M. L. Alterations of deoxyribonucleoside triphosphate pools in Escherichia coli: effects on deoxyribonucleic acid replication and evidence for compartmentation. J Bacteriol. 1979 Nov;140(2):518–524. doi: 10.1128/jb.140.2.518-524.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Pearson G. D., Hanawalt P. C. Isolation of DNA replication complexes from uninfected and adenovirus-infected HeLa cells. J Mol Biol. 1971 Nov 28;62(1):65–80. doi: 10.1016/0022-2836(71)90131-8. [DOI] [PubMed] [Google Scholar]
  35. Reddy G. P., Mathews C. K. Functional compartmentation of DNA precursors in T4 phage-infected bacteria. J Biol Chem. 1978 May 25;253(10):3461–3467. [PubMed] [Google Scholar]
  36. Reddy G. P., Singh A., Stafford M. E., Mathews C. K. Enzyme associations in T4 phage DNA precursor synthesis. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3152–3156. doi: 10.1073/pnas.74.8.3152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Schneider E. L., Stanbridge E. J., Epstein C. J. Incorporation of 3H-uridine and 3H-uracil into RNA: a simple technique for the detection of mycoplasma contamination of cultured cells. Exp Cell Res. 1974 Mar 15;84(1):311–318. doi: 10.1016/0014-4827(74)90411-x. [DOI] [PubMed] [Google Scholar]
  38. Shearman C. W., Kalf G. F. DNA replication by a membrane-DNA complex from rat liver mitochondria. Arch Biochem Biophys. 1977 Aug;182(2):573–586. doi: 10.1016/0003-9861(77)90539-2. [DOI] [PubMed] [Google Scholar]
  39. Shoaf W. T., Jones M. E. Uridylic acid synthesis in Ehrlich ascites carcinoma. Properties, subcellular distribution, and nature of enzyme complexes of the six biosynthetic enzymes. Biochemistry. 1973 Oct 9;12(21):4039–4051. doi: 10.1021/bi00745a004. [DOI] [PubMed] [Google Scholar]
  40. Tomich P. K., Chiu C. S., Wovcha M. G., Greenberg G. R. Evidence for a complex regulating the in vivo activities of early enzymes induced by bacteriophage T4. J Biol Chem. 1974 Dec 10;249(23):7613–7622. [PubMed] [Google Scholar]
  41. Walters R. A., Tobey R. A., Ratliff R. L. Cell-cycle-dependent variations of deoxyribonucleoside triphosphate pools in Chinese hamster cells. Biochim Biophys Acta. 1973 Sep 7;319(3):336–347. doi: 10.1016/0005-2787(73)90173-1. [DOI] [PubMed] [Google Scholar]
  42. Weinstein B. I., Bhardwaj N., Li H. C. A rapid and accurate procedure for assaying DNA polymerase, RNA polymerase, or ribosome dependent protein synthesis. Anal Biochem. 1975 Sep;68(1):62–69. doi: 10.1016/0003-2697(75)90679-x. [DOI] [PubMed] [Google Scholar]
  43. Welch G. R., Gaertner F. H. Influence of an aggregated multienzyme system on transient time: kinetic evidence for compartmentation by an aromatic-amino-acid synthesizing complex of Neurospora crassa. Proc Natl Acad Sci U S A. 1975 Nov;72(11):4218–4222. doi: 10.1073/pnas.72.11.4218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Yamada M., Hanaoka F. Periodic changes in the association of mammalian DNA with the membrane during the cell cycle. Nat New Biol. 1973 Jun 20;243(129):227–230. doi: 10.1038/newbio243227a0. [DOI] [PubMed] [Google Scholar]

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