Reparative Chromatin Assembly Plays an Important Role in Genome Stability

Cover Page

Cite item

Full Text

Open Access Open Access
Restricted Access Access granted
Restricted Access Subscription Access

Abstract

When DNA repair is completed, the processes associated with the restoration of the normal chromatin structure play an important role. Incorrect chromatin assembly can lead to genomic rearrangements, which, in turn, can cause the development of many diseases, including cancer. Previously, we showed that violations of the correct assembly of nucleosomes and their remodulation during the reparative assembly of chromatin lead to an increased level of mutagenesis. In this work, we have shown that the asf1Δ mutation has a constitutively hyperactivated Rad53 kinase, which causes disorganization of the chromatin structure and significantly changes the spectrum of spontaneous reparative mutations. Violation of the binding site of the Rad9 adaptive protein to DNA as a result of inactivation of the DOT1 gene eliminates hif1Δ-specific mutagenesis, which is a consequence of incorrect reparative assembly of nucleosomes. The absence of the Rad9 protein under normal growth conditions and when treated with low doses of UV rays leads to aberrant activation of the RNR complex. At the same time, a further increase in the dose of UV radiation practically does not affect the expression of RNR3. These results confirm that correct chromatin assembly is critical for the normal functioning of the genome.

Full Text

Restricted Access

About the authors

I. I. Skobeleva

Petersburg Nuclear Physics Institute Named by B.P. Konstantinov of National Research Centre “Kurchatov Institute”

Author for correspondence.
Email: alekseeva_ea@pnpi.nrcki.ru
Russian Federation, Gatchina, 188300

T. A. Evstyukhina

Petersburg Nuclear Physics Institute Named by B.P. Konstantinov of National Research Centre “Kurchatov Institute”; Kurchatov Genome Center — Petersburg Nuclear Physics Institute

Email: alekseeva_ea@pnpi.nrcki.ru
Russian Federation, Gatchina, 188300; Gatchina, 188300

E. A. Alekseeva

Petersburg Nuclear Physics Institute Named by B.P. Konstantinov of National Research Centre “Kurchatov Institute”; Kurchatov Genome Center — Petersburg Nuclear Physics Institute

Email: alekseeva_ea@pnpi.nrcki.ru
Russian Federation, Gatchina, 188300; Gatchina, 188300

A. V. Toroshchina

Petersburg Nuclear Physics Institute Named by B.P. Konstantinov of National Research Centre “Kurchatov Institute”

Email: alekseeva_ea@pnpi.nrcki.ru
Russian Federation, Gatchina, 188300

V. T. Peshekhonov

Petersburg Nuclear Physics Institute Named by B.P. Konstantinov of National Research Centre “Kurchatov Institute”; Kurchatov Genome Center — Petersburg Nuclear Physics Institute

Email: alekseeva_ea@pnpi.nrcki.ru
Russian Federation, Gatchina, 188300; Gatchina, 188300

D. V. Fedorov

Petersburg Nuclear Physics Institute Named by B.P. Konstantinov of National Research Centre “Kurchatov Institute”

Email: alekseeva_ea@pnpi.nrcki.ru
Russian Federation, Gatchina, 188300

V. G. Korolev

Petersburg Nuclear Physics Institute Named by B.P. Konstantinov of National Research Centre “Kurchatov Institute”; Kurchatov Genome Center — Petersburg Nuclear Physics Institute

Email: alekseeva_ea@pnpi.nrcki.ru
Russian Federation, Gatchina, 188300; Gatchina, 188300

References

  1. Evstyukhina T.A., Alekseeva E.A., Fedorov D.V. et al. Genetic analysis of the Hsm3 protein function in yeast Saccharomyces cerevisiae NuB4 complex // Genes. 2021. V. 12. P. 1083. https://doi.org/10.3390/ genes12071083
  2. Alekseeva E.A., Evstyukhina T.A., Peshekhonov V.T. et al. The role of the RPD3 complex of Saccharomyces cerevisiae yeast in the activation of UV-induced expression of RNR complex genes // J. Biomed. Res. Environ. Sc. 2024. V. 5. P. 360–372. https://doi.org/10.37871/jbres1902
  3. Alekseeva E.A., Evstyukhina T.A., Peshekhonov V.T., Korolev V.G. Participation of the HIM1 gene of yeast Saccharomyces cerevisiae in the error-free branch of post-replicative repair and role Polη in him1-dependent mutagenesis // Curr Genet. 2021. V. 67. P. 141–151. https://doi.org/10.1007/s00294-020-01115-6
  4. Evstyukhina T.A., Alekseeva E.A., Peshekhonov V.T. et al. The role of chromatin assembly factors in induced mutagenesis at low levels of DNA damage // Genes. 2023. V. 14. P. 1242. https://doi.org/10.3390/genes14061242
  5. Евстюхина Т.А., Алексеева Е.А., Скобелева И.И. и др. Роль различных субъединиц ремоделирующего комплекса INO80 в репарационной сборке хроматина у дрожжей Saccharomyces cerevisiae // Генетика. 2024. Т. 60. № 7. P. 000.
  6. Weinert T.A., Hartwell L.H. The RAD9 gene controls the cycle response to DNA damage checkpoint in Saccharomyces cerevisiae // Science. 1988. V. 241. P. 317–322. https://doi.org/10.1126/science.3291120
  7. Vialarg J.E., Gilbert C.S., Green C.M., Lowndes N.F. The budding yeast Rad9 checkpoint protein is subjected to Mec1/Tel1-dependent hyperphosphorylation and interacts with Rad53 after DNA damage // EMBO J. 1998. V. 17. P. 5679–5688. https://doi.org/10.1093/emboj/17.19.5679
  8. Granata M., Lazzaro F., Novarina D. et al. Dynamics of Rad9 chromatin binding and checkpoint function are mediated by its demirization and are cell cycle-regulated by CDK1 activity // PLoS Genetics. 2010. V. 6. P. e1001047. doi:10.1371/ journal.gen.1001047
  9. Sun Z., Hsiao J., Fay D.S., Stern D.F. Rad53 FHA domain associated with phosphorylated Rad9 in the DNA damage checkpoint // Science. 1998. V. 281. P. 272–274. https://doi.org/10.1126/science.281.5374.272
  10. Wysocki R., Javaheri A., Allard S. et al. Role of Dot1-dependent histone 3 methylation in G, and S phase DNA damage checkpoint functions of Rad9 // Mol. Cell. Biol. 2005. V. 25. P. 8430–8443. https://doi.org/10.1128/MCB.25.19.8430-8443.2005
  11. Javaheri A., Wisocki R., Jobin-Robitaille O. et al. Yeast G1 DNA damage checkpoint regulation by H2A phosphorylation is independent of chromatin remodeling // PNAS. 2006. V. 12. P. 13771–13776. doi/10.1073/pnas.0511192103
  12. Захаров И.А., Кожин С.А., Кожина Т.А. и др. Сборник методик по генетике дрожжей-сахаромицетов, изд. 2-е. Л.: Наука, 1984. 144 с.
  13. Keck K.M., Pemberton L.F. Histone chaperon link histone nuclear import and chromatin assembly // Biochim. Biophys. Acta. 2012. V. 1819. P. 277–289. https://doi.org/10.1016/j.bbagrm.2011.09.007
  14. Adkins M.W., Williams S.K., Linger J., Tyler J.K. Chromatin disassembly from the PHO5 promoter is essential for the recruitment of the general transcription machinery and coactivators // Mol. Cell. Biol. 2007. V. 27. P. 6372–6382. https://doi.org/10.1128/MCB.00981-07
  15. Mousson F., Ochsenbein F., Mann C. The histone chaperone Asf1 at the crossroads of chromatin and DNA checkpoint pathways // Chromosoma. 2007. V. 116. P. 79–93. https://doi.org/10.1007/s00412-006-0087-z
  16. Tyler J.K., Adams C.R., Chen S.R. et al. The RCAF complex mediates chromatin assembly during DNA replication and repair // Nature. 1999. V. 402. P. 555–560. https://doi.org/10.1038/990147
  17. Prado F., Cortes-Ledesma F., Aguilera A. The absence of the yeast chromatin assembly factor Asf1 increases genomic instability and sister chromatid exchange // EMBO Rep. 2004. V. 5. P. 497–502. https://doi.org/10.1038/sj.embor.7400128
  18. Myung K., Pennaneach V., Kats E.S., Kolodner R.D. Saccharomyces cerevisiae chromatin-assembly factors that act during DNA replication function in the maintenance of genome stability // Proc. Natl Acad. Sci. USA. 2003. V. 100. P. 6640–6645. https://doi.org/10.1073/pnas.1232239100
  19. Kozmin S.G., Pavlov Y.I., Kunkel T.A., Sage E. Roles of Saccharomyces cerevisiae DNA polymerases Polη and Polζ in response to irradiation by simulated sunlight // Nucl. Acids Res. 2003. V. 31. P. 4541–4552. https://doi.org/10.1093/nar/gkg489
  20. Кожина Т.Н., Евстюхина Т.А., Пешехонов В.Т. и др. Гистон-метилазы Dot1 и Set2 контролируют уровень спонтанного и УФ-индуцированного мутагенеза в дрожжах Saccharomyces cerevisiae // Генетика. 2016. Т. 52. № 3. С. 300–310.
  21. Sharp J.A., Fouts E.T., Krawitz D.C., Kaufman P.D. Yeast histone deposition protein Asf1p requires Hir proteins and PCNA for heterochromatic silencing // Curr Biol. 2001. V. 11. P. 463–473. https://doi.org/10.1016/s0960-9822(01)00140-3
  22. Ge Z., Wang H., Parthun M.R. Nuclear Hat1p complex (NuB4) components participate in DNA repair-linked chromatin reassembly // J. Biol. Chem. 2011. V. 286. P. 16790–16799. https://doi.org/10.1074/jbc.M110.216846
  23. Bostelman L.J., Keller A.M., Albrecht A.M. et al. Methylation of histone H3 lysine-79 by Dot1p plays multiple roles in the response to UV damage in Saccharomyces cerevisiae // DNA Repair. 2007. V. 6. P. 383–395. https://doi.org/10.1016/j.dnarep.2006.12.010
  24. Conde F., San-Segundo P.A. Role of Dot1 in the response to alkylating DNA damage in Saccharomyces cerevisiae: Regulation of DNA damage tolerance by the error-prone polymerases Polzeta/Rev1 // Genetics. 2008. V. 179. P. 1197–1210. https://doi.org/10.1534/genetics.108.089003

Supplementary files

Supplementary Files
Action
1. JATS XML
Download
2. 1. Relative normalized expression of the RNR3 gene in various mutant strains. The histogram shows mutant strains hat1Δ and asf1Δ before and after irradiation with ultraviolet light (after UV irradiation, the cells were kept for four hours at 30 ° C in an induction thermostat), the dose of UV radiation was 256 J/m2; *p < 0.05, Student's t-criterion.

Download (111KB)
Indexing metadata
3. 2. Inactivation of the RAD9 gene does not affect the frequency of UV-induced mutagenesis at the CAN 1 locus at high doses of UV. The graph shows the frequency of mutagenesis in the wild-type strain and the mutant rad9Δ strain when irradiated with doses of 14, 28, 42, 84 and 126 J/m2 of ultraviolet radiation.Figure 2-6 shows the average values and standard errors of the average value (± SEM) obtained as a result of five independent experiments.

Download (76KB)
Indexing metadata
4. 3. Inactivation of the RAD9 or DOT1 genes leads to a decrease in the frequency of UV-induced mutagenesis at the CAN1 locus at low UV doses and increased sensitivity to UV light of the rad9Δ strain.The graphs show the wild-type strain and mutant strains rad9Δ and dot1Δ when irradiated with doses of 7, 14 and 21 J/m2 of ultraviolet radiation.

Download (127KB)
Indexing metadata
5. 4. Inactivation of the HIM1 or HIF1 genes in the dot1Δ strain affects the sensitivity to UV light and the frequency of UV-induced mutagenesis at the CAN1 locus at low UV doses.The graphs show the wild-type strain and mutant strains dot1Δ, hif1Δ, him1Δ, dot1Δ him1Δ and dot1Δ hif1Δ when irradiated with doses of 7, 14 and 21 J/m2 of ultraviolet radiation.

Download (175KB)
Indexing metadata
6. 5. Inactivation of the IES5 or NHP10 genes in the dot1Δ strain affects the sensitivity to UV light and the frequency of UV-induced mutagenesis at the CAN1 locus at low UV doses.The graphs show the wild-type strain and mutant strains ies5Δ, nhp10Δ, dot1Δ ies5Δ and dot1Δ nhp10Δ when irradiated with doses of 7, 14 and 21 J/m2 of ultraviolet radiation.

Download (171KB)
Indexing metadata
7. 6. Inactivation of the NHP10 gene in the red53+HA-A strain does not affect sensitivity to UV light and leads to a decrease in the frequency of UV-induced mutagenesis at the CAN1 locus at low UV doses. The graphs show the wild-type strain and mutant strains nhp10Δ, rad53+HA-F and nhp10Δ rad53+HA-F when irradiated with doses of 7, 14 and 21 J/m2 of ultraviolet radiation.

Download (173KB)
Indexing metadata
8. 7. Relative normalized expression of the RNR3 gene in various mutant strains. The histogram shows the wild-type strain and mutant strains rad9Δ, dot1Δ and dot1Δ nhp10Δ before and after irradiation with ultraviolet light (after UV irradiation, the cells were kept for four hours at 30 °C in an induction thermostat), the dose of UV radiation was 14 and 256 J/m2; *p < 0.05, Student's t-test.

Download (75KB)
Indexing metadata

Copyright (c) 2025 Russian Academy of Sciences