scholarly journals Saccharomyces cerevisiae H2A copies differentially contribute to recombination and CAG/CTG repeat maintenance, with a role for H2A.1 threonine 126

2018 ◽  
Author(s):  
Nealia C.M. House ◽  
Erica J. Polleys ◽  
Ishtiaque Quasem ◽  
Cailin E. Joyce ◽  
Oliver Takacsi-Nagy ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Dna Repair ◽  
Homologous Recombination ◽  
Cag Repeat ◽  
Break Induced Replication ◽  
Sister Chromatid Recombination ◽  
True Homolog ◽  
Ctg Repeat

AbstractDNA are sites of genomic instability. Long CAG/CTG repeats form hairpin structures, are fragile, and can expand during DNA repair. The chromatin response to DNA damage can influence repair fidelity, but the knowledge of chromatin modifications involved in maintaining repair fidelity within repetitive DNA is limited. In a screen for CAG repeat fragility in Saccharomyces cerevisiae, histone 2A copy 1 (H2A.1) was identified to protect the repeat from increased rates of breakage. To address the role of H2A in CAG repeat instability, we tested the effect of deleting each histone H2 subytpe. Whereas deletion of HTA2, HTZ1, HTB1, and HTB2 did not significantly affect CAG repeat maintenance, deletion of HTA1 resulted in increased expansion frequency. Notably, mutation of threonine 126, unique to H2A.1, to a non-phosphorylatable alanine increased CAG repeat instability to a similar level as the hta1Δ mutant. CAG instability in the absence of HTA1 or mutation to hta1-T126A was dependent on the presence of the homologous recombination (HR) repair proteins Rad51, Rad52, and Rad57, and the Polδ subunit Pol32. In addition, sister chromatid recombination (SCR) was suppressed in the hta1Δ and hta1-T126A mutants and this suppression was epistatic to pol32Δ. Finally, break-induced replication (BIR) is impaired in the hta1Δ mutant, resulting in an altered repair profile. These data reveal differential roles for the H2A subtypes in DNA repair and implicate a new role for H2A.1 threonine-126 phosphorylation in mediating fidelity during HR repair and promoting SCR. Using a fragile, repetive DNA element to model endogenous DNA damage, our results demonstrate that H2A.1 plays a greater role than H2A.2 in promoting homology-dependent repair, suggesting H2A.1 is the true homolog of mammalian H2AX, whereas H2A.2 is functionally equivalent to mammalian H2A.Author SummaryCAG/CTG trinuncleotide repeats are fragile sequences that when expanded can cause human disease. To evaluate the role of S. cerevisiae histone H2A copies in DNA repair, we have measured instability of an expanded CAG/CTG repeat tract and repair outcomes in H2A mutants. Although the two copies of H2A are nearly identical in amino acid sequence, we found that the CAG repeat is more unstable in the absence of H2A copy 1 (H2A.1) than H2A copy 2, and that this role appears to be partially dependent on a phosphorylatable threonine at residue 126 in the C-terminal tail of H2A.1. Further, we show through a series of genetic assays that H2A.1 plays a role in promoting homologous recombination events, including sister chromatid recombination and break-induced replication. Our results uncover a role for H2A.1 in mediating fidelity of repair within repetitive DNA, and demonstrate that modification of its unique Thr126 residue plays a role in regulating SCR. Given the dependence of HR repair on H2A.1 but not H2A.2, we conclude that H2A.1 plays a greater repair-specific role in the cell and therefore would be the true homolog of mammalian H2AX.

scholarly journals The Role of the Rad55–Rad57 Complex in DNA Repair

Genes ◽  
2021 ◽  
Vol 12 (9) ◽  
pp. 1390
Author(s):  
Upasana Roy ◽  
Eric C. Greene
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Repair ◽  
Homologous Recombination ◽  
Single Molecule ◽  
Genome Integrity ◽  
Genetic Studies ◽  
Biochemical Assays ◽  
Rad51 Paralogs ◽  
Higher Eukaryotes

Homologous recombination (HR) is a mechanism conserved from bacteria to humans essential for the accurate repair of DNA double-stranded breaks, and maintenance of genome integrity. In eukaryotes, the key DNA transactions in HR are catalyzed by the Rad51 recombinase, assisted by a host of regulatory factors including mediators such as Rad52 and Rad51 paralogs. Rad51 paralogs play a crucial role in regulating proper levels of HR, and mutations in the human counterparts have been associated with diseases such as cancer and Fanconi Anemia. In this review, we focus on the Saccharomyces cerevisiae Rad51 paralog complex Rad55–Rad57, which has served as a model for understanding the conserved role of Rad51 paralogs in higher eukaryotes. Here, we discuss the results from early genetic studies, biochemical assays, and new single-molecule observations that have together contributed to our current understanding of the molecular role of Rad55–Rad57 in HR.

scholarly journals Distinct roles for S. cerevisiae H2A copies in recombination and repeat stability, with a role for H2A.1 threonine 126

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Nealia CM House ◽  
Erica J Polleys ◽  
Ishtiaque Quasem ◽  
Marjorie De la Rosa Mejia ◽  
Cailin E Joyce ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Cag Repeat ◽  
High Fidelity ◽  
Specific Function ◽  
D Loop ◽  
Dna Secondary Structures ◽  
Break Induced Replication ◽  
Sister Chromatid Recombination ◽  
Ctg Repeat ◽  
Expanded Form

CAG/CTG trinuncleotide repeats are fragile sequences that when expanded form DNA secondary structures and cause human disease. We evaluated CAG/CTG repeat stability and repair outcomes in histone H2 mutants in S. cerevisiae. Although the two copies of H2A are nearly identical in amino acid sequence, CAG repeat stability depends on H2A copy 1 (H2A.1) but not copy 2 (H2A.2). H2A.1 promotes high-fidelity homologous recombination, sister chromatid recombination (SCR), and break-induced replication whereas H2A.2 does not share these functions. Both decreased SCR and the increase in CAG expansions were due to the unique Thr126 residue in H2A.1 and hta1Δ or hta1-T126A mutants were epistatic to deletion of the Polδ subunit Pol32, suggesting a role for H2A.1 in D-loop extension. We conclude that H2A.1 plays a greater repair-specific role compared to H2A.2 and may be a first step towards evolution of a repair-specific function for H2AX compared to H2A in mammalian cells.

Saccharomyces cerevisiae rad51 Mutants Are Defective in DNA Damage-Associated Sister Chromatid Exchanges but Exhibit Increased Rates of Homology-Directed Translocations

Genetics ◽  
2001 ◽  
Vol 158 (3) ◽  
pp. 959-972
Author(s):  
Michael Fasullo ◽  
Peter Giallanza ◽  
Zheng Dong ◽  
Cinzia Cera ◽  
Thomas Bennett
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Sister Chromatid ◽  
Sister Chromatid Exchanges ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
Dna Damaging Agents ◽  
Break Induced Replication ◽  
Chromatid Exchanges

Abstract Saccharomyces cerevisiae Rad51 is structurally similar to Escherichia coli RecA. We investigated the role of S. cerevisiae RAD51 in DNA damage-associated unequal sister chromatid exchanges (SCEs), translocations, and inversions. The frequency of these rearrangements was measured by monitoring mitotic recombination between two his3 fragments, his3-Δ5′ and his3-Δ3′::HOcs, when positioned on different chromosomes or in tandem and oriented in direct or inverted orientation. Recombination was measured after cells were exposed to chemical agents and radiation and after HO endonuclease digestion at his3-Δ3′::HOcs. Wild-type and rad51 mutant strains showed no difference in the rate of spontaneous SCEs; however, the rate of spontaneous inversions was decreased threefold in the rad51 mutant. The rad51 null mutant was defective in DNA damage-associated SCE when cells were exposed to either radiation or chemical DNA-damaging agents or when HO endonuclease-induced double-strand breaks (DSBs) were directly targeted at his3-Δ3′::HOcs. The defect in DNA damage-associated SCEs in rad51 mutants correlated with an eightfold higher spontaneous level of directed translocations in diploid strains and with a higher level of radiation-associated translocations. We suggest that S. cerevisiae RAD51 facilitates genomic stability by reducing nonreciprocal translocations generated by RAD51-independent break-induced replication (BIR) mechanisms.

scholarly journals RNase H enables efficient repair of R-loop induced DNA damage

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Jeremy D Amon ◽  
Douglas Koshland
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Dna Repair ◽  
Genome Instability ◽  
Rnase H ◽  
Rna Polymerase I ◽  
Chromosomal Dna ◽  
Polymerase I ◽  
Break Induced Replication ◽  
Kinetics Of

R-loops, three-stranded structures that form when transcripts hybridize to chromosomal DNA, are potent agents of genome instability. This instability has been explained by the ability of R-loops to induce DNA damage. Here, we show that persistent R-loops also compromise DNA repair. Depleting endogenous RNase H activity impairs R-loop removal in Saccharomyces cerevisiae, causing DNA damage that occurs preferentially in the repetitive ribosomal DNA locus (rDNA). We analyzed the repair kinetics of this damage and identified mutants that modulate repair. We present a model that the persistence of R-loops at sites of DNA damage induces repair by break-induced replication (BIR). This R-loop induced BIR is particularly susceptible to the formation of lethal repair intermediates at the rDNA because of a barrier imposed by RNA polymerase I.

scholarly journals A Role for Histone H2B During Repair of UV-Induced DNA Damage in Saccharomyces cerevisiae

Genetics ◽  
2002 ◽  
Vol 160 (4) ◽  
pp. 1375-1387
Author(s):  
Emmanuelle M D Martini ◽  
Scott Keeney ◽  
Mary Ann Osley
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Chromatin Structure ◽  
Specific Effect ◽  
Cell Killing ◽  
Cyclobutane Pyrimidine Dimers ◽  
Histone H2b ◽  
Uv Sensitivity ◽  
Pyrimidine Dimers

Abstract To investigate the role of the nucleosome during repair of DNA damage in yeast, we screened for histone H2B mutants that were sensitive to UV irradiation. We have isolated a new mutant, htb1-3, that shows preferential sensitivity to UV-C. There is no detectable difference in bulk chromatin structure or in the number of UV-induced cis-syn cyclobutane pyrimidine dimers (CPD) between HTB1 and htb1-3 strains. These results suggest a specific effect of this histone H2B mutation in UV-induced DNA repair processes rather than a global effect on chromatin structure. We analyzed the UV sensitivity of double mutants that contained the htb1-3 mutation and mutations in genes from each of the three epistasis groups of RAD genes. The htb1-3 mutation enhanced UV-induced cell killing in rad1Δ and rad52Δ mutants but not in rad6Δ or rad18Δ mutants, which are defective in postreplicational DNA repair (PRR). When combined with other mutations that affect PRR, the histone mutation increased the UV sensitivity of strains with defects in either the error-prone (rev1Δ) or error-free (rad30Δ) branches of PRR, but did not enhance the UV sensitivity of a strain with a rad5Δ mutation. When combined with a ubc13Δ mutation, which is also epistatic with rad5Δ, the htb1-3 mutation enhanced UV-induced cell killing. These results suggest that histone H2B acts in a novel RAD5-dependent branch of PRR.

scholarly journals DNA Damage Response in Multiple Myeloma: The Role of the Tumor Microenvironment

Cancers ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 504
Author(s):  
Takayuki Saitoh ◽  
Tsukasa Oda
Keyword(s):  
Multiple Myeloma ◽  
Dna Damage ◽  
Dna Repair ◽  
Tumor Microenvironment ◽  
Dna Damage Response ◽  
Genetic Instability ◽  
Dna Repair Mechanisms ◽  
Damage Response ◽  
Repair Mechanisms

Multiple myeloma (MM) is an incurable plasma cell malignancy characterized by genomic instability. MM cells present various forms of genetic instability, including chromosomal instability, microsatellite instability, and base-pair alterations, as well as changes in chromosome number. The tumor microenvironment and an abnormal DNA repair function affect genetic instability in this disease. In addition, states of the tumor microenvironment itself, such as inflammation and hypoxia, influence the DNA damage response, which includes DNA repair mechanisms, cell cycle checkpoints, and apoptotic pathways. Unrepaired DNA damage in tumor cells has been shown to exacerbate genomic instability and aberrant features that enable MM progression and drug resistance. This review provides an overview of the DNA repair pathways, with a special focus on their function in MM, and discusses the role of the tumor microenvironment in governing DNA repair mechanisms.

scholarly journals Effect of the expression of BRCA2 on spontaneous homologous recombination and DNA damage-induced nuclear foci in Saccharomyces cerevisiae

Mutagenesis ◽  
2013 ◽  
Vol 28 (2) ◽  
pp. 187-195 ◽  
Author(s):  
L. Spugnesi ◽  
C. Balia ◽  
A. Collavoli ◽  
E. Falaschi ◽  
V. Quercioli ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Homologous Recombination ◽  
Nuclear Foci

scholarly journals The Role of Posttranslational Modifications in DNA Repair

2020 ◽  
Vol 2020 ◽  
pp. 1-13
Author(s):  
Miaomiao Bai ◽  
Dongdong Ti ◽  
Qian Mei ◽  
Jiejie Liu ◽  
Xin Yan ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Genome Stability ◽  
Protein Localization ◽  
Complex Structure ◽  
Protein Protein Interactions ◽  
Regulate Protein

The human body is a complex structure of cells, which are exposed to many types of stress. Cells must utilize various mechanisms to protect their DNA from damage caused by metabolic and external sources to maintain genomic integrity and homeostasis and to prevent the development of cancer. DNA damage inevitably occurs regardless of physiological or abnormal conditions. In response to DNA damage, signaling pathways are activated to repair the damaged DNA or to induce cell apoptosis. During the process, posttranslational modifications (PTMs) can be used to modulate enzymatic activities and regulate protein stability, protein localization, and protein-protein interactions. Thus, PTMs in DNA repair should be studied. In this review, we will focus on the current understanding of the phosphorylation, poly(ADP-ribosyl)ation, ubiquitination, SUMOylation, acetylation, and methylation of six typical PTMs and summarize PTMs of the key proteins in DNA repair, providing important insight into the role of PTMs in the maintenance of genome stability and contributing to reveal new and selective therapeutic approaches to target cancers.

scholarly journals Regulation of the cognitive aging process by the transcriptional repressor RP58

2021 ◽  
Author(s):  
Tomoko Tanaka ◽  
Shinobu Hirai ◽  
Hiroyuki Manabe ◽  
Kentaro Endo ◽  
Hiroko Shimbo ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Knockout Mice ◽  
Cognitive Aging ◽  
Critical Role ◽  
Harmful Effect ◽  
Transcriptional Repressor ◽  
Repair Pathway ◽  
Wild Type

Aging involves a decline in physiology which is a natural event in all living organisms. An accumulation of DNA damage contributes to the progression of aging. DNA is continually damaged by exogenous sources and endogenous sources. If the DNA repair pathway operates normally, DNA damage is not life threatening. However, impairments of the DNA repair pathway may result in an accumulation of DNA damage, which has a harmful effect on health and causes an onset of pathology. RP58, a zinc-finger transcriptional repressor, plays a critical role in cerebral cortex formation. Recently, it has been reported that the expression level of RP58 decreases in the aged human cortex. Furthermore, the role of RP58 in DNA damage is inferred by the involvement of DNMT3, which acts as a co-repressor for RP58, in DNA damage. Therefore, RP58 may play a crucial role in the DNA damage associated with aging. In the present study, we investigated the role of RP58 in aging. We used RP58 hetero-knockout and wild-type mice in adolescence, adulthood, or old age. We performed immunohistochemistry to determine whether microglia and DNA damage markers responded to the decline in RP58 levels. Furthermore, we performed an object location test to measure cognitive function, which decline with age. We found that the wild-type mice showed an increase in single-stranded DNA and gamma-H2AX foci. These results indicate an increase in DNA damage or dysfunction of DNA repair mechanisms in the hippocampus as age-related changes. Furthermore, we found that, with advancing age, both the wild-type and hetero-knockout mice showed an impairment of spatial memory for the object and increase in reactive microglia in the hippocampus. However, the RP58 hetero-knockout mice showed these symptoms earlier than the wild-type mice did. These results suggest that a decline in RP58 level may lead to the progression of aging.

scholarly journals Arabidopsis Mediator subunit 17 connects transcription with DNA repair after UV-B exposure

2021 ◽  
Author(s):  
Marisol Giustozzi ◽  
Santiago Freytes ◽  
Aime Jaskolowski ◽  
Micaela Lichy ◽  
Julieta L. Mateos ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Transcription Initiation ◽  
Plant Responses ◽  
Repair System ◽  
Root Tips ◽  
Direct Role ◽  
Altered Expression ◽  
Eukaryotic Organisms

Mediator 17 (MED17) is a subunit of the Mediator complex that regulates transcription initiation in eukaryotic organisms. In yeast and humans, MED17 also participates in DNA repair, physically interacting with proteins of the Nucleotide Excision DNA Repair system. We here analyzed the role of MED17 in Arabidopsis plants exposed to UV-B radiation, which role has not been previously described. Comparison of med17 mutant transcriptome to that of WT plants showed that almost one third of transcripts with altered expression in med17 plants are also changed by UV-B exposure in WT plants. To validate the role of MED17 in UV-B irradiated plants, plant responses to UV-B were analyzed, including flowering time, DNA damage accumulation and programmed cell death in the meristematic cells of the root tips. Our results show that med17 and OE MED17 plants have altered responses to UV-B; and that MED17 participates in various aspects of the DNA damage response (DDR). Increased sensitivity to DDR after UV-B in med17 plants can be due to altered regulation of UV-B responsive transcripts; but additionally MED17 physically interacts with DNA repair proteins, suggesting a direct role of this Mediator subunit during repair. Finally, we here also show that MED17 is necessary to regulate the DDR activated by ATR, and that PDCD5 overexpression reverts the deficiencies in DDR shown in med17 mutants. Together, the data presented demonstrates that MED17 is an important regulator of the DDR after UV-B radiation in Arabidopsis plants.

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