Characteristics of replication-independent endogenous double-strand breaks in Saccharomyces cerevisiae

Pongpanich, Monnat; Patchsung, Maturada; Thongsroy, Jirapan; Mutirangura, Apiwat

doi:10.1186/1471-2164-15-750

Cited by 12 publications

(34 citation statements)

References 21 publications

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“…Second, we observed accumulation of replication-independent endogenous DNA double strand breaks (RIND-EDSBs) in hypermethylate genome [42]. Our recent study indicated that RIND-EDSBs may possess an epigenetic role in reducing DNA strain similar to a small gap between two railway lines [43][44][45]. Third, DNA repair activity is different depending on chromatin structure [46,47].…”

Section: Discussionmentioning

confidence: 90%

Alu siRNA to Increase Alu Element Methylation and Prevent DNA Damage

et al. 2018

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Global DNA hypomethylation promoting genomic instability leads to cancer and deterioration of human health with age. Aim: To invent a biotechnology that can reprogram this process. Methods: We used Alu siRNA to direct Alu interspersed repetitive sequences methylation in human cells. We evaluated the correlation between DNA damage and Alu methylation levels. Results: We observed an inverse correlation between Alu element methylation and endogenous DNA damage in white blood cells. Cells transfected with Alu siRNA exhibited high Alu methylation levels, increased proliferation, reduced endogenous DNA damage and improved resistance to DNA damaging agents. Conclusion: Alu methylation stabilizes the genome by preventing accumulation of DNA damage. Alu siRNA could be useful for evaluating reprograming of the global hypomethylation phenotype in cancer and aging cells. DNA methylation at interspersed repetitive sequences (IRSs) plays an important role in maintaining genome stability. Cells with IRS hypomethylation exhibit increased mutation rates [1,2]. Here, we tested whether DNA damage, an alteration in the chemical structure of DNA and a precursor to mutation [3], plays a role in mediating how global hypomethylation promotes genomic instability. Our recent study found that global hypomethylation is associated with plasma 8-hydroxy-2 -deoxyguanosine (8-OHdG) in biliary atresia patients [4]. Moreover, urinary 8-OHdG, DNA strand breaks and global DNA hypomethylation are associated with low serum folate [5] and oxidative stress [6]. Therefore, we hypothesized that the human genome may use DNA methylation in IRSs to prevent DNA damage.This study developed a technology to add DNA methylation at Alu elements. The human genome contains greater than one million Alu elements [7]. Several reports demonstrated de novo methylation by siRNA in plants [8][9][10]. In human cells, small RNA was used to promote DNA methylation, shRNA for long interspersed element-1 (LINE-1) and hepatitis B virus, and siRNA for HIV-1 promoter region [11][12][13]. There are many types of IRS such as Alu elements, LINE-1, and human endogenous retrovirus [14][15][16][17]. To increase DNA methylation, we tested siRNA to unique sequences and several IRS sequences. Our preliminary trial demonstrated that only Alu siRNA could increase methylation of the target sequences. Here, we evaluated whether Alu siRNA is a useful tool to explore the role of global hypomethylation in genomic instability [18].Alu hypomethylation may play a role in causing genomic instability in cancer and aging cells. Genomic instability, the main enabling characteristic of cancer and aging processes [18,19], may mainly be promoted by IRS hypomethylation. IRS hypomethylation is commonly observed both in aging [14,20] and cancer cells [21]. IRS

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Section: Discussionmentioning

confidence: 90%

Alu siRNA to Increase Alu Element Methylation and Prevent DNA Damage

et al. 2018

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“…Instead, they may possess important biological functions, perhaps in relieving topological stress that might otherwise result in uncontrolled DNA breakage. They have also been reported to occur nonrandomly, for example, with an increased frequency in heterochromatin (200). Such breaks are repaired by either Ku-or Rad51p-dependent pathways, as evidenced by the increased levels of such breaks in cells deleted for either Ku or Rad51p (201).…”

Section: Hmo1 and Dna Double-strand Break Repairmentioning

confidence: 97%

Yeast HMO1: Linker Histone Reinvented

Panday

Grove

2017

Microbiol Mol Biol Rev

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SUMMARY Eukaryotic genomes are packaged in chromatin. The higher-order organization of nucleosome core particles is controlled by the association of the intervening linker DNA with either the linker histone H1 or high mobility group box (HMGB) proteins. While H1 is thought to stabilize the nucleosome by preventing DNA unwrapping, the DNA bending imposed by HMGB may propagate to the nucleosome to destabilize chromatin. For metazoan H1, chromatin compaction requires its lysine-rich C-terminal domain, a domain that is buried between globular domains in the previously characterized yeast Saccharomyces cerevisiae linker histone Hho1p. Here, we discuss the functions of S. cerevisiae HMO1, an HMGB family protein unique in containing a terminal lysine-rich domain and in stabilizing genomic DNA. On ribosomal DNA (rDNA) and genes encoding ribosomal proteins, HMO1 appears to exert its role primarily by stabilizing nucleosome-free regions or “fragile” nucleosomes. During replication, HMO1 likewise appears to ensure low nucleosome density at DNA junctions associated with the DNA damage response or the need for topoisomerases to resolve catenanes. Notably, HMO1 shares with the mammalian linker histone H1 the ability to stabilize chromatin, as evidenced by the absence of HMO1 creating a more dynamic chromatin environment that is more sensitive to nuclease digestion and in which chromatin-remodeling events associated with DNA double-strand break repair occur faster; such chromatin stabilization requires the lysine-rich extension of HMO1. Thus, HMO1 appears to have evolved a unique linker histone-like function involving the ability to stabilize both conventional nucleosome arrays as well as DNA regions characterized by low nucleosome density or the presence of noncanonical nucleosomes.

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“…The yeast strains used in this study were obtained from the Haber Laboratory (Charlestown, MA, USA) (37) and included JKM115 [MAT-a DhohmlD::ADE1 () hmrD::ADE1 lys5 leu2 ura3 trp1], JKM179 [MAT-a DhohmlD::ADE1 hmrD::ADE1 lys5 leu2 ura3 trp1 ade3::GALHO (HO nuclease gene regulated by the GAL1 promoter)], BY4741, ypr052cΔ (nhp6aΔ), and YAA25 (+ HO, mec1D sml1D), as previously described (14,15). For chronological aging experiments, yeast cells were grown in yeast peptone (YP) medium + 2% glucose at 30°C on a rotary shaker at 250 rpm overnight and then switched to YP medium + 2% raffinose for 2 d before being washed and resuspended in sterile deionized water.…”

Section: Yeast Strains Media and Growth Conditionsmentioning

confidence: 99%

“…As previously described (15), ligated HMW DNA was digested with RsaI and ligated again with the second linkers. The HMW DNA was subjected to PCR with 2 primers of each linker.…”

Section: Library Preparation and Sequencingmentioning

confidence: 99%

“…Physiological RIND-EDSBs display many characteristics of epigenetic markers and share no similarities with DNA lesions, whereas pathological DSBs must be immediately repaired to prevent apoptotic signaling (12). By contrast, RIND-EDSBs are evolutionarily conserved in eukaryotes and are retained in cells that have been exposed to neither radiation nor DNA-damaging agents, even in the presence of functional DSB repair mechanisms (7,(13)(14)(15). RIND-EDSBs are localized within the hypermethylated genome (7,13); however, endogenous g-H2A histone family member X (H2AX) deposition on chromatin is more prevalent in hypomethylated DNA (13).…”

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Reduction in replication‐independent endogenous DNA double‐strand breaks promotes genomic instability during chronological aging in yeast

et al. 2018

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The mechanism that causes genomic instability in nondividing aging cells is unknown. Our previous study of mutant yeast suggested that 2 types of replication-independent endogenous DNA double-strand breaks (RIND-EDSBs) exist and that they play opposing roles. The first type, known as physiologic RIND-EDSBs, were ubiquitous in the G phase of both yeast and human cells in certain genomic locations and may act as epigenetic markers. Low RIND-EDSB levels were found in mutants that lacked chromatin-condensing proteins, such as the high-mobility group box (HMGB) proteins and Sir2. The second type is referred to as pathologic RIND-EDSBs. High pathological RIND-EDSB levels were found in DSB repair mutants. Under normal physiologic conditions, these excess RIND-EDSBs are repaired in much the same way as DNA lesions. Here, chronological aging in yeast reduced physiological RIND-EDSBs and cell viability. A strong correlation was observed between the reduction in RIND-EDSBs and viability in aging yeast cells ( r = 0.94, P < 0.0001). We used galactose-inducible HO endonuclease (HO) and nhp6a∆, an HMGB protein mutant, to evaluate the consequences of reduced physiological RIND-EDSB levels. The HO-induced cells exhibited a sustained reduction in RIND-EDSBs at various levels for several days. Interestingly, we found that lower physiologic RIND-EDSB levels resulted in decreased cell viability ( r = 0.69, P < 0.0001). Treatment with caffeine, a DSB repair inhibitor, increased pathological RIND-EDSBs, which were distinguished from physiologic RIND-EDSBs by their lack of sequences prior to DSB in untreated cells [odds ratio (OR) ≤1]. Caffeine treatment in both the HO-induced and nhp6a∆ cells markedly increased OR ≤1 breaks. Therefore, physiological RIND-EDSBs play an epigenetic role in preventing pathological RIND-EDSBs, a type of DNA damage. In summary, the reduction of physiological RIND-EDSB level is a genomic instability mechanism in chronologically aging cells.-Thongsroy, J., Patchsung, M., Pongpanich, M., Settayanon, S., Mutirangura, A. Reduction in replication-independent endogenous DNA double-strand breaks promotes genomic instability during chronological aging in yeast.

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Characteristics of replication-independent endogenous double-strand breaks in Saccharomyces cerevisiae

Cited by 12 publications

References 21 publications

Alu siRNA to Increase Alu Element Methylation and Prevent DNA Damage

Alu siRNA to Increase Alu Element Methylation and Prevent DNA Damage

Yeast HMO1: Linker Histone Reinvented

Reduction in replication‐independent endogenous DNA double‐strand breaks promotes genomic instability during chronological aging in yeast

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