Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1997 Mar 1;25(5):1056–1063. doi: 10.1093/nar/25.5.1056

Enzymatic activities involved in the DNA resynthesis step of nucleotide excision repair are firmly attached to chromatin.

K Bouayadi 1, A van der Leer-van Hoffen 1, A S Balajee 1, A T Natarajan 1, A A van Zeeland 1, L H Mullenders 1
PMCID: PMC146546  PMID: 9023118

Abstract

In this study the role of nuclear architecture in nucleotide excision repair (NER) was investigated by gentle dismantling of the cell and probing the capability of chromatin to carry out repair in vitro. The rationale behind this approach is that compartmentalization of NER at nuclear structures would make the enzymatic activities refractory to extraction by buffers that solubilize cellular membranes. In order to obtain intact chromatin primary human fibroblasts were encapsulated in agarose microbeads and lysed in isotonic buffers containing the non-ionic detergent Triton X-100. Under these conditions the majority of cellular proteins diffuse out of the beads, but the remaining chromatin is able to replicate and to transcribe DNA in the presence of triphosphates and Mg2+. UV irradiation of confluent repair-proficient human fibroblasts prior to lysis stimulated the incorporation of deoxynucleotide triphosphates in Triton X-100-isolated chromatin, even under stringent lysis conditions. In addition, experiments with UV-sensitive xeroderma pigmentosum (complementation groups A and C) and Cockayne's syndrome fibroblasts (complementation group A) revealed that this repair synthesis was due to global genome repair activity. Transcription-coupled repair was only detectable in cells permeabilized by streptolysin O (SLO). Repair synthesis in Triton X-100-isolated chromatin amounted to 15% of the total repair synthesis as measured in SLO-permeabilized cells. To allow the detection of these activities in vitro, presynthesis complexes have to be formed in intact cells, indicating that chromatin from Triton X-100-lysed cells is unable to initiate NER in vitro. Our data indicate that the components involved in the resynthesis step of NER are tightly associated with chromatin. A substantial fraction of total proliferating cell nuclear antigen (PCNA), which is required for the resynthesis step in NER, has been reported to become Triton X-100 non-extractable and tightly associated with nuclear structures after UV irradiation of cells. We propose that Triton X-100-resistant repair synthesis might be mediated by this chromatin-bound fraction of total PCNA.

Full Text

The Full Text of this article is available as a PDF (120.7 KB).

Selected References

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

  1. Aboussekhra A., Wood R. D. Detection of nucleotide excision repair incisions in human fibroblasts by immunostaining for PCNA. Exp Cell Res. 1995 Dec;221(2):326–332. doi: 10.1006/excr.1995.1382. [DOI] [PubMed] [Google Scholar]
  2. Bravo R., Macdonald-Bravo H. Existence of two populations of cyclin/proliferating cell nuclear antigen during the cell cycle: association with DNA replication sites. J Cell Biol. 1987 Oct;105(4):1549–1554. doi: 10.1083/jcb.105.4.1549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ciejek E. M., Tsai M. J., O'Malley B. W. Actively transcribed genes are associated with the nuclear matrix. Nature. 1983 Dec 8;306(5943):607–609. doi: 10.1038/306607a0. [DOI] [PubMed] [Google Scholar]
  4. Dickinson P., Cook P. R., Jackson D. A. Active RNA polymerase I is fixed within the nucleus of HeLa cells. EMBO J. 1990 Jul;9(7):2207–2214. doi: 10.1002/j.1460-2075.1990.tb07390.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Hanawalt P. C. DNA repair comes of age. Mutat Res. 1995 Mar;336(2):101–113. doi: 10.1016/0921-8777(94)00061-a. [DOI] [PubMed] [Google Scholar]
  6. Huang S., Spector D. L. Nascent pre-mRNA transcripts are associated with nuclear regions enriched in splicing factors. Genes Dev. 1991 Dec;5(12A):2288–2302. doi: 10.1101/gad.5.12a.2288. [DOI] [PubMed] [Google Scholar]
  7. Jackson D. A., Balajee A. S., Mullenders L., Cook P. R. Sites in human nuclei where DNA damaged by ultraviolet light is repaired: visualization and localization relative to the nucleoskeleton. J Cell Sci. 1994 Jul;107(Pt 7):1745–1752. doi: 10.1242/jcs.107.7.1745. [DOI] [PubMed] [Google Scholar]
  8. Jackson D. A., Cook P. R. A general method for preparing chromatin containing intact DNA. EMBO J. 1985 Apr;4(4):913–918. doi: 10.1002/j.1460-2075.1985.tb03718.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Jackson D. A., Cook P. R. Transcription occurs at a nucleoskeleton. EMBO J. 1985 Apr;4(4):919–925. doi: 10.1002/j.1460-2075.1985.tb03719.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jackson D. A., Cook P. R. Transcription occurs at a nucleoskeleton. EMBO J. 1985 Apr;4(4):919–925. doi: 10.1002/j.1460-2075.1985.tb03719.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jackson D. A., Hassan A. B., Errington R. J., Cook P. R. Visualization of focal sites of transcription within human nuclei. EMBO J. 1993 Mar;12(3):1059–1065. doi: 10.1002/j.1460-2075.1993.tb05747.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jackson D. A., Yuan J., Cook P. R. A gentle method for preparing cyto- and nucleo-skeletons and associated chromatin. J Cell Sci. 1988 Jul;90(Pt 3):365–378. doi: 10.1242/jcs.90.3.365. [DOI] [PubMed] [Google Scholar]
  13. Jenkins H., Hölman T., Lyon C., Lane B., Stick R., Hutchison C. Nuclei that lack a lamina accumulate karyophilic proteins and assemble a nuclear matrix. J Cell Sci. 1993 Sep;106(Pt 1):275–285. doi: 10.1242/jcs.106.1.275. [DOI] [PubMed] [Google Scholar]
  14. Keeney S., Linn S. A critical review of permeabilized cell systems for studying mammalian DNA repair. Mutat Res. 1990 Sep-Nov;236(2-3):239–252. doi: 10.1016/0921-8777(90)90008-s. [DOI] [PubMed] [Google Scholar]
  15. Miura M., Domon M., Sasaki T., Kondo S., Takasaki Y. Two types of proliferating cell nuclear antigen (PCNA) complex formation in quiescent normal and xeroderma pigmentosum group A fibroblasts following ultraviolet light (uv) irradiation. Exp Cell Res. 1992 Aug;201(2):541–544. doi: 10.1016/0014-4827(92)90308-u. [DOI] [PubMed] [Google Scholar]
  16. Miura M., Domon M., Sasaki T., Takasaki Y. Induction of proliferating cell nuclear antigen (PCNA) complex formation in quiescent fibroblasts from a xeroderma pigmentosum patient. J Cell Physiol. 1992 Feb;150(2):370–376. doi: 10.1002/jcp.1041500221. [DOI] [PubMed] [Google Scholar]
  17. Mullenders L. H., Vrieling H., Venema J., van Zeeland A. A. Hierarchies of DNA repair in mammalian cells: biological consequences. Mutat Res. 1991 Sep-Oct;250(1-2):223–228. doi: 10.1016/0027-5107(91)90179-r. [DOI] [PubMed] [Google Scholar]
  18. Mullenders L. H., van Kesteren van Leeuwen A. C., van Zeeland A. A., Natarajan A. T. Nuclear matrix associated DNA is preferentially repaired in normal human fibroblasts, exposed to a low dose of ultraviolet light but not in Cockayne's syndrome fibroblasts. Nucleic Acids Res. 1988 Nov 25;16(22):10607–10622. doi: 10.1093/nar/16.22.10607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mullenders L. H., van Kesteren A. C., Bussmann C. J., van Zeeland A. A., Natarajan A. T. Distribution of u.v.-induced repair events in higher-order chromatin loops in human and hamster fibroblasts. Carcinogenesis. 1986 Jun;7(6):995–1002. doi: 10.1093/carcin/7.6.995. [DOI] [PubMed] [Google Scholar]
  20. Nakayasu H., Berezney R. Mapping replicational sites in the eucaryotic cell nucleus. J Cell Biol. 1989 Jan;108(1):1–11. doi: 10.1083/jcb.108.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pagano M., Theodoras A. M., Tam S. W., Draetta G. F. Cyclin D1-mediated inhibition of repair and replicative DNA synthesis in human fibroblasts. Genes Dev. 1994 Jul 15;8(14):1627–1639. doi: 10.1101/gad.8.14.1627. [DOI] [PubMed] [Google Scholar]
  22. Park M. S., Knauf J. A., Pendergrass S. H., Coulon C. H., Strniste G. F., Marrone B. L., MacInnes M. A. Ultraviolet-induced movement of the human DNA repair protein, Xeroderma pigmentosum type G, in the nucleus. Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8368–8373. doi: 10.1073/pnas.93.16.8368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Schaeffer L., Roy R., Humbert S., Moncollin V., Vermeulen W., Hoeijmakers J. H., Chambon P., Egly J. M. DNA repair helicase: a component of BTF2 (TFIIH) basic transcription factor. Science. 1993 Apr 2;260(5104):58–63. doi: 10.1126/science.8465201. [DOI] [PubMed] [Google Scholar]
  24. Shivji K. K., Kenny M. K., Wood R. D. Proliferating cell nuclear antigen is required for DNA excision repair. Cell. 1992 Apr 17;69(2):367–374. doi: 10.1016/0092-8674(92)90416-a. [DOI] [PubMed] [Google Scholar]
  25. Smith C. A., Hanawalt P. C. Phage T4 endonuclease V stimulates DNA repair replication in isolated nuclei from ultraviolet-irradiated human cells, including xeroderma pigmentosum fibroblasts. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2598–2602. doi: 10.1073/pnas.75.6.2598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Stief A., Winter D. M., Strätling W. H., Sippel A. E. A nuclear DNA attachment element mediates elevated and position-independent gene activity. Nature. 1989 Sep 28;341(6240):343–345. doi: 10.1038/341343a0. [DOI] [PubMed] [Google Scholar]
  27. Sugasawa K., Masutani C., Hanaoka F. Cell-free repair of UV-damaged simian virus 40 chromosomes in human cell extracts. I. Development of a cell-free system detecting excision repair of UV-irradiated SV40 chromosomes. J Biol Chem. 1993 Apr 25;268(12):9098–9104. [PubMed] [Google Scholar]
  28. Toschi L., Bravo R. Changes in cyclin/proliferating cell nuclear antigen distribution during DNA repair synthesis. J Cell Biol. 1988 Nov;107(5):1623–1628. doi: 10.1083/jcb.107.5.1623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Vaughn J. P., Dijkwel P. A., Mullenders L. H., Hamlin J. L. Replication forks are associated with the nuclear matrix. Nucleic Acids Res. 1990 Apr 25;18(8):1965–1969. doi: 10.1093/nar/18.8.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Venema J., Bartosová Z., Natarajan A. T., van Zeeland A. A., Mullenders L. H. Transcription affects the rate but not the extent of repair of cyclobutane pyrimidine dimers in the human adenosine deaminase gene. J Biol Chem. 1992 May 5;267(13):8852–8856. [PubMed] [Google Scholar]
  31. Venema J., van Hoffen A., Karcagi V., Natarajan A. T., van Zeeland A. A., Mullenders L. H. Xeroderma pigmentosum complementation group C cells remove pyrimidine dimers selectively from the transcribed strand of active genes. Mol Cell Biol. 1991 Aug;11(8):4128–4134. doi: 10.1128/mcb.11.8.4128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Xing Y., Johnson C. V., Dobner P. R., Lawrence J. B. Higher level organization of individual gene transcription and RNA splicing. Science. 1993 Feb 26;259(5099):1326–1330. doi: 10.1126/science.8446901. [DOI] [PubMed] [Google Scholar]
  33. van Hoffen A., Natarajan A. T., Mayne L. V., van Zeeland A. A., Mullenders L. H., Venema J. Deficient repair of the transcribed strand of active genes in Cockayne's syndrome cells. Nucleic Acids Res. 1993 Dec 25;21(25):5890–5895. doi: 10.1093/nar/21.25.5890. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. van Zeeland A. A., Smith C. A., Hanawalt P. C. Sensitive determination of pyrimidine dimers in DNA of UV-irradiated mammalian cells. Introduction of T4 endonuclease V into frozen and thawed cells. Mutat Res. 1981 Jun;82(1):173–189. doi: 10.1016/0027-5107(81)90148-2. [DOI] [PubMed] [Google Scholar]

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

RESOURCES