Reparative Chromatin Assembly Plays an Important Role in Genome Stability

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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.

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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

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Supplementary files

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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