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Detection of Alkali-Labile Sites on Satellite DNA by DNA Breakage Coupled with Fluorescence in Situ Hybridization (DNA-FISH) Monitor DNA Damage in Cervical Epithelial Cells

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Abstract

Satellite DNA is the main component of functional centromeres and forms the main structural constituent of heterochromatin. The presence of alkali-labile sites (ALSs) is a feature inherent to chromatin structure. Here we aimed to characterize ALSs in different satellite DNA loci in cervical epithelial cells using DNA breakage detection coupled with fluorescence in situ hybridization (DBD–FISH). Cervical epithelial cells embedded in an agarose matrix were deproteinized and exposed to alkaline denaturation, which generated single-stranded DNA (ssDNA) starting from the ends of spontaneous basal DNA breaks and ALSs. The amount of ssDNA produced within a specific sequence area could be detected by DBD–FISH using specific probes. The DBD–FISH signals, which were corrected for respective FISH signals during metaphase, were remarkably stronger in the 5 bp classical satellite DNA domains analyzed (D1Z1, D9Z3, and D16Z3) compared with alphoid satellite regions (D3Z1, D8Z2, and DXZ1). D1Z1 locus of chromome-1 being the most affected by alkali denaturation, and contrary, D3Z1 locus of chromosome–3 was the least sensitive to alkali treatment. These findings suggest a high density of constitutive ALSs—probably abasic sites—within the 5 bp satellite DNA sequences in cervical epithelial cells. The presence and relative abundance of ALSs might help explain the high frequency of spontaneous breakage and rearrangements in the pericentromeric heterochromatin of chromosomes 1, 9, and 16 when this chromatin region is undercondensed spontaneously or via induction, such as following viral infections. ALSs in these sequences could be useful tools to monitor DNA damage in cases of cervical carcinogenesis. ALSs on these sequences could be useful tools to monitor DNA damage in cases of cervical carcinogenesis.

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REFERENCES

  1. Tyler-Smith, C. and Willard, H.F., Mammalian chromosome structure, Curr. Opin. Genet. Dev., 1993, vol. 3, no. 3, pp. 390–397. https://doi.org/10.1016/0959-437x(93)90110-b

    Article  CAS  PubMed  Google Scholar 

  2. Meyne, J., Goodwin, E.H., and Moyzis, R.K., Chromosome localization and orientation of the simple sequence repeat of human satellite I DNA, Chromosoma, 1994, vol. 103, no. 2, pp. 99–103. https://doi.org/10.1007/bf00352318

    Article  CAS  PubMed  Google Scholar 

  3. Albright, K.L., Bartholdi, M.F., Cram, L.S., Deaven, L.L., Hildebrand, C.E., Joste, E.N., Longmire, J.L., Meyne, J., and Schwarzacher-Robinson, T., Human chromosome-specific repetitive DNA sequences: novel markers for genetic analysis, Chromosoma, 1987, vol. 95, no. 6, pp. 375–386. https://doi.org/10.1007/bf00333988

    Article  PubMed  Google Scholar 

  4. Jeanpierre, M., Human satellite 2 and 3, Ann. Génét., 1994, vol. 37, no. 4, pp. 163–171.

    CAS  PubMed  Google Scholar 

  5. Aze, A., Sannino, V., Soffientini, P., Bachi, A., and Costanzo, V., Centromeric DNA replication reconstitution reveals DNA loops and ATR checkpoint suppression, Nat. Cell Biol., 2016, vol. 18, no. 6, pp. 8684–8691. https://doi.org/10.1038/ncb3344

    Article  CAS  Google Scholar 

  6. Fernández, J.L., Goyanes, V., Ramiro-Diaz, J., and Gosálvez, J., Application of FISH for in situ detection and quantification of DNA breaks, Cytogenet. Cell Genet., 1998, vol. 82, nos 3-4, pp. 251–256. https://doi.org/10.1159/000015112

    Article  PubMed  Google Scholar 

  7. Cortés-Gutiérrez, E.I., Dávila-Rodríguez, M.I., Fernandez, J.L., López-Fernández, C., and Gosálvez, J., DNA damage in women with cervical neoplasia evaluated by DNA breakage detection-fluorescence in situ hybridization, Anal. Quant. Cytol. Histol., 2011, vol. 33, no. 3, pp. 175–181.

    PubMed  Google Scholar 

  8. Vogt, P., Code domains in tandem repetitive DNA sequence structures, Chromosoma, 1992, vol. 101, no. 10, pp. 585–589. https://doi.org/10.1007/bf00360534

    Article  CAS  PubMed  Google Scholar 

  9. Fernández, J.L., Valverde, D., Goyanes, V., Buño, I., and Gosálvez, J., Alu I in situ digestion of human alphoid and classical satellite DNA regions: high-resolution digital image analysis of FISH signals from condensed and extended chromatin, Cytogenet. Cell Genet., 1997, vol. 76, nos. 1–2, pp. 94–100. https://doi.org/10.1159/000134522

    Article  PubMed  Google Scholar 

  10. Gosálvez, J., López-Fernández, C., Goyanes, V., and Mezzanotte, R., Chromosome differentiation using nucleases: an overview, in Chromosomes Today, Henriques-Gil, N., Parker, J.S., and Puertas, M.J., Eds., London: Chapman and Hall, 1997, vol. 12, pp. 23–49. https://doi.org/10.1007/978-94-009-1537-4_2

    Google Scholar 

  11. Darzynkiewicz, Z., Huang, X., and Okafuji, M., Detection of DNA strand breaks by flow and laser scanning cytometry in studies of apoptosis and cell proliferation (DNA replication), Methods Mol. Biol., 2006, vol. 314, pp. 81–93. https://doi.org/10.1385/1-59259-973-7:081

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Gore, J., Bryant, Z., Nollmann, M., Le, M.U., Cozzarelli, N.R., and Bustamante, C., DNA overwinds when stretched, Nature, 2006, vol. 442, no. 7104, pp. 836–839. https://doi.org/10.1038/nature04974

    Article  CAS  PubMed  Google Scholar 

  13. Fernández, J.L., Vázquez-Gundín, F., Rivero, M.T., Genescá, A., Gosálvez, J., and Goyanes, V., DBD-FISH on neutral comets: simultaneous analysis of DNA single- and double-strand breaks in individual cells, Exp. Cell Res., 2001, vol. 270, no. 1, pp. 102–109. https://doi.org/10.1007/978-1-60327-409-8_11

    Article  CAS  PubMed  Google Scholar 

  14. Cortés-Gutiérrez, E.I., Ortíz-Hernández, B.L., Dávila-Rodríguez, M.I., Cerda-Flores, R.M., Fernández, J.L., López-Fernández, C., and Gosálvez, J., 5-bp Classical satellite DNA loci from chromosome-1 instability in cervical neoplasia detected by DNA breakage detection/fluorescence in situ hybridization (DBD-FISH), Int. J. Mol. Sci., 2013, vol. 14, no. 2, pp. 4135–4147. https://doi.org/10.3390/ijms14024135

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Cortés-Gutiérrez, E.I., Dávila-Rodríguez, M.I., Fernández, J.L., López-Fernández, C., and Gosálvez, J., Koilocytes are enriched for alkaline-labile sites, Eur. J. Histochem., 2010, vol. 54, no. 4. e32. https://doi.org/10.4081/ejh.2010.e32

    Article  PubMed  PubMed Central  Google Scholar 

  16. Fernández, J.L., Vazquez-Gundin, F., Rivero, M.T., Goyanes, V., and Gosálvez, J., Evidence of abundant constitutive alkali-labile sites in human 5 bp classical satellite DNA loci by DBD-FISH, Mutat. Res., 2001, vol. 473, no. 2, pp. 163–168. https://doi.org/10.1016/s0027-5107(00)00146-9

    Article  PubMed  Google Scholar 

  17. Cortés-Gutiérrez, E.I., Dávila-Rodríguez, M.I., López-Fernández, C., Fernández, J.L., and Gosálvez, J., Alkali-labile sites in sperm cells from Sus and Ovis species, Int. J. Androl., 2008, vol. 31, no. 3, pp. 354–363. https://doi.org/10.1111/j.1365-2605.2007.00781.x

    Article  PubMed  Google Scholar 

  18. Vazquez-Gundin, F., Goyanes, V., Campos, A., Blasco, M., Gosálvez, J., and Fernández, J.L., High frequency of constitutive alkali-labile sites in mouse major satellite DNA, detected by DNA breakage detection-fluorescence in situ hybridization, Mutat. Res., 2001, vol. 483, nos. 1–2, pp. 43–50. https://doi.org/10.1016/s0027-5107(01)00218-4

    Article  PubMed  Google Scholar 

  19. Rivero, M.T., Mosquera, A., Goyanes, V., Slijepcevic, P., and Fernández, J.L., Differences in repair profiles of interstitial telomeric sites between normal and DNA double-strand ALS in mammalian sperm break repair deficient Chinese hamster cells, Exp. Cell Res., 2004, vol. 295, no. 1, pp. 161–172. https://doi.org/10.1016/j.yexcr.2003.12.031

    Article  CAS  PubMed  Google Scholar 

  20. López-Fernández, C., Arroyo, F., Fernández, J.L., and Gosálvez, J., Interstitial telomeric sequence blocks in constitutive pericentromeric heterochromatin from Pyrgomorpha conica (Orthoptera) are enriched in constitutive alkali-labile sites, Mutat. Res., 2006, vol. 599, nos. 1–2, pp. 36–44. https://doi.org/10.1016/j.mrfmmm.2006.01.004

    Article  CAS  PubMed  Google Scholar 

  21. Cortés-Gutiérrez, E.I., López-Fernández, C., Fernández, J.L., Dávila-Rodríguez, M.I., Johnston, S.D., and Gosálvez, J., Interpreting sperm DNA damage in a diverse range of mammalian sperm by means of the two-tailed comet assay, Front. Genet., 2014, vol. 27, no. 5, p. 404. https://doi.org/10.3389/fgene.2014.00404

    Article  CAS  Google Scholar 

  22. Von Sonntag, C., Carbohydrate radicals: from ethylene glycol to DNA strand breakage, Int. J. Radiat. Biol., 2014, vol. 90, no. 6, pp. 416–422. https://doi.org/10.3109/09553002.2014.908040

    Article  CAS  PubMed  Google Scholar 

  23. Bandyopadhyay, R., Berend, S.A., Page, S.L., Choo, K.H., and Shaffer, L.G., Satellite III sequences on 14p and their relevance to Robertsonian translocation formation, Chromosome Res., 2001, vol. 9, no. 3, pp. 235–242. https://doi.org/10.1023/a:1016652621226

    Article  CAS  PubMed  Google Scholar 

  24. Lana, E., Megarbane, A., Tourriere, H., Sarda, P., Lefranc, G., Claustres, M., and De Sario, A., DNA replication is altered in Immunodeficiency Centromeric instability Facial anomalies (ICF) cells carrying DNMT3B mutations, Eur. J. Hum. Genet., 2012, vol. 20, no. 1, pp. 1044–1050. https://doi.org/10.1038/ejhg.2012.41

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Jefferson, A., Colella, S., Moralli, D., Wilson, N., Yusuf, M., Gimelli, G., Ragoussis, J., and Volpi, E.V., Altered intra-nuclear organisation of heterochromatin and genes in ICF syndrome, PLoS One, 2010, vol. 5, no. 6. e11 364. https://doi.org/10.1371/journal.pone.0011364

    Article  CAS  Google Scholar 

  26. Toubiana, S., Velasco, G., Chityat, A., Kaindl, A.M., Hershtig, N., Tzur-Gilat, A., and Francastel, C., Subtelomeric methylation distinguishes between subtypes of Immunodeficiency. Centromeric Instability and Facial Anomalies (ICF) syndrome, Hum. Mol. Genet., 2018, vol. 27, no. 20, pp. 3568–3581. https://doi.org/10.1093/hmg/ddy265

    Article  CAS  PubMed  Google Scholar 

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Funding

This study was supported by Fondo de Investigation en Salud-IMSS (FIS/IMSS/PROT/MD13/1247 and Fondo Sectorial de Investigación en Salud y Seguridad Social SS/IMSS/ISSSTE-CONACYT (293539).

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Authors and Affiliations

  1. Department of Genetics, Centro de Investigación Biomédica del Noreste, Instituto Mexicano del Seguro Social, IMSS, 64720, Monterrey, México

    C. Garcia-Vielma & M. I. Dávila-Rodriguez

  2. Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León, Monterrey, México

    E. I. Cortés-Gutiérrez & Juan Antonio Garcia Salas

Authors
  1. C. Garcia-Vielma
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  2. E. I. Cortés-Gutiérrez
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  3. Juan Antonio Garcia Salas
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  4. M. I. Dávila-Rodriguez
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Correspondence to E. I. Cortés-Gutiérrez.

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Conflict of interest. The authors declare that they have no conflict of interest.

Statement of compliance with standards of research involving humans as subjects. The study was approved by the local Centro de Investigatión Biomédica del Noreste (CIBIN), IMSS Ethics Committee. All procedures performed in studies involving human participants were in accordance with the ethical standards of the University of British Columbia and with the 1964 Declaration of Helsinki and its later amendments. Informed consent was obtained from all individual participants included in the study.

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Garcia-Vielma, C., Cortés-Gutiérrez, E.I., Garcia Salas, J.A. et al. Detection of Alkali-Labile Sites on Satellite DNA by DNA Breakage Coupled with Fluorescence in Situ Hybridization (DNA-FISH) Monitor DNA Damage in Cervical Epithelial Cells. Cytol. Genet. 54, 173–178 (2020). https://doi.org/10.3103/S0095452720020061

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