Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1983 Jul 25;11(14):4793–4807. doi: 10.1093/nar/11.14.4793

Identification of proteins interacting with newly replicated DNA in SV40-infected cells by UV-induced DNA-protein cross-linking.

K Tsutsui, S Watanabe, M Katagiri, T Oda
PMCID: PMC326086  PMID: 6308560

Abstract

Protein species interacting with newly replicated DNA were analyzed using a photo cross-linking technique. Nascent DNA was labeled in vitro with [alpha-32P]dCTP and BrdUTP in SV40-infected CV-1 cells made permeable with saponin. The labeled cells were then irradiated with UV light (254 nm) and were treated extensively with DNase I. Proteins with radioactive DNA tags were separated by SDS-PAGE and visualized by autoradiography. Among 10-15 proteins which were cross-linked, the proteins with apparent molecular weights of 16.5 K, 44 K, 82 K and those in the 94-140 K region appeared to be associated with newly replicated SV40 DNA. A pulse-chase experiment showed that the 82 K and 94-140 K proteins interacted with new DNA in a relatively localized region close to the replication fork. The 44 K protein was identified as the major viral capsid protein, VP1, using antiserum to SV40 capsid proteins. It was suggested that VP1 binds to nascent DNA shortly after DNA synthesis and migrates into chromatin maturation regions.

Full text

PDF
4793

Images in this article

4799Image
on p.4799
4799Image
on p.4799
4800Image
on p.4800
4803Image
on p.4803

Selected References

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

  1. Annunziato A. T., Schindler R. K., Riggs M. G., Seale R. L. Association of newly synthesized histones with replicating and nonreplicating regions of chromatin. J Biol Chem. 1982 Jul 25;257(14):8507–8515. [PubMed] [Google Scholar]
  2. Annunziato A. T., Schindler R. K., Thomas C. A., Jr, Seale R. L. Dual nature of newly replicated chromatin. Evidence for nucleosomal and non-nucleosomal DNA at the site of native replication forks. J Biol Chem. 1981 Nov 25;256(22):11880–11886. [PubMed] [Google Scholar]
  3. Brady J. N., Consigli R. A. Chromatographic separation of the polyoma virus proteins and renaturation of the isolated VP1 major capsid protein. J Virol. 1978 Aug;27(2):436–442. doi: 10.1128/jvi.27.2.436-442.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cao T. M., Sung M. T. Ultraviolet light induced preferential cross-linking of histone H3 to deoxyribonucleic acid in chromatin and nuclei of chicken erythrocytes. Biochemistry. 1982 Jul 6;21(14):3419–3427. doi: 10.1021/bi00257a027. [DOI] [PubMed] [Google Scholar]
  5. Cusick M. E., Herman T. M., DePamphilis M. L., Wassarman P. M. Structure of chromatin at deoxyribonucleic acid replication forks: prenucleosomal deoxyribonucleic acid is rapidly excised from replicating simian virus 40 chromosomes by micrococcal nuclease. Biochemistry. 1981 Nov 10;20(23):6648–6658. doi: 10.1021/bi00526a020. [DOI] [PubMed] [Google Scholar]
  6. DePamphilis M. L., Wassarman P. M. Replication of eukaryotic chromosomes: a close-up of the replication fork. Annu Rev Biochem. 1980;49:627–666. doi: 10.1146/annurev.bi.49.070180.003211. [DOI] [PubMed] [Google Scholar]
  7. Eriksson S., Caras I. W., Martin D. W., Jr Direct photoaffinity labeling of an allosteric site on subunit protein M1 of mouse ribonucleotide reductase by dTTP. Proc Natl Acad Sci U S A. 1982 Jan;79(1):81–85. doi: 10.1073/pnas.79.1.81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Harrison C. A., Turner D. H., Hinkle D. C. Laser crosslinking of E. coli RNA polymerase and T7 DNA. Nucleic Acids Res. 1982 Apr 10;10(7):2399–2414. doi: 10.1093/nar/10.7.2399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Herman T. M., DePamphilis M. L., Wassarman P. M. Structure of chromatin at deoxyribonucleic acid replication forks: Okazaki fragments released from replicating SV40 chromosomes by single-strand specific endonucleases are not in nucleosomes. Biochemistry. 1979 Oct 16;18(21):4563–4571. doi: 10.1021/bi00588a017. [DOI] [PubMed] [Google Scholar]
  10. Herman T. M., DePamphilis M. L., Wassarman P. M. Structure of chromatin at deoxyribonucleic acid replication forks: location of the first nucleosomes on newly synthesized simian virus 40 deoxyribonucleic acid. Biochemistry. 1981 Feb 3;20(3):621–630. doi: 10.1021/bi00506a027. [DOI] [PubMed] [Google Scholar]
  11. Jackson V., Chalkley R. A new method for the isolation of replicative chromatin: selective deposition of histone on both new and old DNA. Cell. 1981 Jan;23(1):121–134. doi: 10.1016/0092-8674(81)90277-4. [DOI] [PubMed] [Google Scholar]
  12. Krokan H., Schaffer P., DePamphilis M. L. Involvement of eucaryotic deoxyribonucleic acid polymerases alpha and gamma in the replication of cellular and viral deoxyribonucleic acid. Biochemistry. 1979 Oct 2;18(20):4431–4443. doi: 10.1021/bi00587a025. [DOI] [PubMed] [Google Scholar]
  13. Kunkel G. R., Martinson H. G. Histone-DNA interactions within chromatin. Isolation of histones from DNA-histone adducts induced in nuclei by UV light. Nucleic Acids Res. 1978 Nov;5(11):4263–4272. doi: 10.1093/nar/5.11.4263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. Levy A., Jakob K. M. Nascent DNA in nucleosome like structures from chromatin. Cell. 1978 Jun;14(2):259–267. doi: 10.1016/0092-8674(78)90112-5. [DOI] [PubMed] [Google Scholar]
  16. Mandel R., Kolomijtseva G., Brahms J. G. DNA-protein interactions in nucleosomes and in chromatin. Structural studies of chromatin stabilized by ultraviolet-light induced crosslinking. Eur J Biochem. 1979 May 15;96(2):257–265. doi: 10.1111/j.1432-1033.1979.tb13036.x. [DOI] [PubMed] [Google Scholar]
  17. Markovitz A. Ultraviolet light-induced stable complexes of DNA and DNA polymerase. Biochim Biophys Acta. 1972 Nov 9;281(4):522–534. doi: 10.1016/0005-2787(72)90153-0. [DOI] [PubMed] [Google Scholar]
  18. Milavetz B., Hopkins T. Simian virus 40 encapsidation: characterization of early intermediates. J Virol. 1982 Sep;43(3):830–839. doi: 10.1128/jvi.43.3.830-839.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Moyne G., Harper F., Saragosti S., Yaniv M. Absence of nucleosomes in a histone-containing nucleoprotein complex obtained by dissociation of purified SV40 virions. Cell. 1982 Aug;30(1):123–130. doi: 10.1016/0092-8674(82)90018-6. [DOI] [PubMed] [Google Scholar]
  20. Oda T., Watanabe S., Maki Y., Iwamoto H. Nucleotide requirements for DNA replication of SV40 chromatin in digitonin-treated and saponin-treated permeable cells. Nucleic Acids Symp Ser. 1981;(10):159–162. [PubMed] [Google Scholar]
  21. Riley D., Weintraub H. Conservative segregation of parental histones during replication in the presence of cycloheximide. Proc Natl Acad Sci U S A. 1979 Jan;76(1):328–332. doi: 10.1073/pnas.76.1.328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Roufa D. J., Marchionni M. A. Nucleosome segregation at a defined mammalian chromosomal site. Proc Natl Acad Sci U S A. 1982 Mar;79(6):1810–1814. doi: 10.1073/pnas.79.6.1810. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sato K., Hosokawa K. The structure of adenovirion chromatin revealed by ultraviolet light-induced cross-linking. Biochem Biophys Res Commun. 1981 Aug 31;101(4):1318–1323. doi: 10.1016/0006-291x(81)91591-6. [DOI] [PubMed] [Google Scholar]
  24. Schiaffonati L., Tsutsui Y., Chang S. D., Baserga R. Effect of 5-bromodeoxyuridine on deoxyribonucleic acid-protein adducts induced by ultraviolet light on chromatin cells. Lab Invest. 1978 Jan;38(1):58–66. [PubMed] [Google Scholar]
  25. Seidman M. M., Levine A. J., Weintraub H. The asymmetric segregation of parental nucleosomes during chrosome replication. Cell. 1979 Oct;18(2):439–449. doi: 10.1016/0092-8674(79)90063-1. [DOI] [PubMed] [Google Scholar]
  26. Sperling J., Sperling R. Photochemical cross-linking of histones to DNA nucleosomes. Nucleic Acids Res. 1978 Aug;5(8):2755–2773. doi: 10.1093/nar/5.8.2755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Strniste G. F., Rall S. C. Induction of stable protein-deoxyribonucleic acid adducts in Chinese hamster cell chromatin by ultraviolet light. Biochemistry. 1976 Apr 20;15(8):1712–1719. doi: 10.1021/bi00653a019. [DOI] [PubMed] [Google Scholar]
  28. Tapper D. P., DePamphilis M. L. Discontinuous DNA replication: accumulation of Simian virus 40 DNA at specific stages in its replication. J Mol Biol. 1978 Apr 15;120(3):401–422. doi: 10.1016/0022-2836(78)90427-8. [DOI] [PubMed] [Google Scholar]
  29. Worcel A., Han S., Wong M. L. Assembly of newly replicated chromatin. Cell. 1978 Nov;15(3):969–977. doi: 10.1016/0092-8674(78)90280-5. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES