scholarly journals An Assay to Study Intra-Chromosomal Deletions in Yeast

2019 ◽  
Vol 2 (3) ◽  
pp. 74 ◽  
Author(s):  
Bailey E. Lucas ◽  
Matthew T. McPherson ◽  
Tila M. Hawk ◽  
Lexia N. Wilson ◽  
Jacob M. Kroh ◽  
...  
Keyword(s):  
Dna Damage ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Chromosomal Deletions ◽  
Damage Repair ◽  
Damage Response ◽  
Dna Damage Response Pathway ◽  
Genetic Assay

An accurate DNA damage response pathway is critical for the repair of DNA double-strand breaks. Repair may occur by homologous recombination, of which many different sub-pathways have been identified. Some recombination pathways are conservative, meaning that the chromosome sequences are preserved, and others are non-conservative, leading to some alteration of the DNA sequence. We describe an in vivo genetic assay to study non-conservative intra-chromosomal deletions at regions of non-tandem direct repeats in Schizosaccharomyces pombe. This assay can be used to study both spontaneous breaks arising during DNA replication and induced double-strand breaks created with the S. cerevisiae homothallic endonuclease (HO). The preliminary genetic validation of this assay shows that spontaneous breaks require rad52+ but not rad51+, while induced breaks require both genes, in agreement with previous studies. This assay will be useful in the field of DNA damage repair for studying mechanisms of intra-chromosomal deletions.

scholarly journals Schizosaccharomyces pombe Assays to Study Mitotic Recombination Outcomes

Genes ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 79
Author(s):  
Hannah M. Hylton ◽  
Bailey E. Lucas ◽  
Ruben C. Petreaca
Keyword(s):  
Dna Damage ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Molecular Mechanisms ◽  
Mitotic Recombination ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Damage Repair

The fission yeast—Schizosaccharomyces pombe—has emerged as a powerful tractable system for studying DNA damage repair. Over the last few decades, several powerful in vivo genetic assays have been developed to study outcomes of mitotic recombination, the major repair mechanism of DNA double strand breaks and stalled or collapsed DNA replication forks. These assays have significantly increased our understanding of the molecular mechanisms underlying the DNA damage response pathways. Here, we review the assays that have been developed in fission yeast to study mitotic recombination.

scholarly journals RepID-deficient cancer cells are sensitized to a drug targeting p97/VCP segregase

2021 ◽  
Author(s):  
Sang-Min Jang ◽  
Christophe E. Redon ◽  
Haiqing Fu ◽  
Fred E. Indig ◽  
Mirit I. Aladjem
Keyword(s):  
Dna Damage ◽  
Cancer Cells ◽  
Cell Sorting ◽  
Clonogenic Survival ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Ubiquitinated Proteins ◽  
Damage Response ◽  
Survival Assay

Abstract Background The p97/valosin-containing protein (VCP) complex is a crucial factor for the segregation of ubiquitinated proteins in the DNA damage response and repair pathway. Objective We investigated whether blocking the p97/VCP function can inhibit the proliferation of RepID-deficient cancer cells using immunofluorescence, clonogenic survival assay, fluorescence-activated cell sorting, and immunoblotting. Result p97/VCP was recruited to chromatin and colocalized with DNA double-strand breaks in RepID-deficient cancer cells that undergo spontaneous DNA damage. Inhibition of p97/VCP induced death of RepID-depleted cancer cells. This study highlights the potential of targeting p97/VCP complex as an anticancer therapeutic approach. Conclusion Our results show that RepID is required to prevent excessive DNA damage at the endogenous levels. Localization of p97/VCP to DSB sites was induced based on spontaneous DNA damage in RepID-depleted cancer cells. Anticancer drugs targeting p97/VCP may be highly potent in RepID-deficient cells. Therefore, we suggest that p97/VCP inhibitors synergize with RepID depletion to kill cancer cells.

scholarly journals Sae2 antagonizes Rad9 accumulation at DNA double-strand breaks to attenuate checkpoint signaling and facilitate end resection

2018 ◽  
Vol 115 (51) ◽  
pp. E11961-E11969 ◽  
Author(s):  
Tai-Yuan Yu ◽  
Michael T. Kimble ◽  
Lorraine S. Symington
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Inhibitory Effects ◽  
Checkpoint Signaling ◽  
Synthetic Lethal ◽  
Strand Breaks ◽  
Damage Response ◽  
End Resection

The Mre11-Rad50-Xrs2NBS1 complex plays important roles in the DNA damage response by activating the Tel1ATM kinase and catalyzing 5′–3′ resection at DNA double-strand breaks (DSBs). To initiate resection, Mre11 endonuclease nicks the 5′ strands at DSB ends in a reaction stimulated by Sae2CtIP. Accordingly, Mre11-nuclease deficient (mre11-nd) and sae2Δ mutants are expected to exhibit similar phenotypes; however, we found several notable differences. First, sae2Δ cells exhibit greater sensitivity to genotoxins than mre11-nd cells. Second, sae2Δ is synthetic lethal with sgs1Δ, whereas the mre11-nd sgs1Δ mutant is viable. Third, Sae2 attenuates the Tel1-Rad53CHK2 checkpoint and antagonizes Rad953BP1 accumulation at DSBs independent of Mre11 nuclease. We show that Sae2 competes with other Tel1 substrates, thus reducing Rad9 binding to chromatin and to Rad53. We suggest that persistent Sae2 binding at DSBs in the mre11-nd mutant counteracts the inhibitory effects of Rad9 and Rad53 on Exo1 and Dna2-Sgs1–mediated resection, accounting for the different phenotypes conferred by mre11-nd and sae2Δ mutations. Collectively, these data show a resection initiation independent role for Sae2 at DSBs by modulating the DNA damage checkpoint.

scholarly journals Recent advances in the nucleolar responses to DNA double-strand breaks

2020 ◽  
Vol 48 (17) ◽  
pp. 9449-9461
Author(s):  
Lea Milling Korsholm ◽  
Zita Gál ◽  
Blanca Nieto ◽  
Oliver Quevedo ◽  
Stavroula Boukoura ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Damage Response ◽  
Ribosomal Dna ◽  
Repetitive Sequences ◽  
Dna Lesions ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Damage Response

Abstract DNA damage poses a serious threat to human health and cells therefore continuously monitor and repair DNA lesions across the genome. Ribosomal DNA is a genomic domain that represents a particular challenge due to repetitive sequences, high transcriptional activity and its localization in the nucleolus, where the accessibility of DNA repair factors is limited. Recent discoveries have significantly extended our understanding of how cells respond to DNA double-strand breaks (DSBs) in the nucleolus, and new kinases and multiple down-stream targets have been identified. Restructuring of the nucleolus can occur as a consequence of DSBs and new data point to an active regulation of this process, challenging previous views. Furthermore, new insights into coordination of cell cycle phases and ribosomal DNA repair argue against existing concepts. In addition, the importance of nucleolar-DNA damage response (n-DDR) mechanisms for maintenance of genome stability and the potential of such factors as anti-cancer targets is becoming apparent. This review will provide a detailed discussion of recent findings and their implications for our understanding of the n-DDR. The n-DDR shares features with the DNA damage response (DDR) elsewhere in the genome but is also emerging as an independent response unique to ribosomal DNA and the nucleolus.

scholarly journals βarrestin-1 regulates DNA repair by acting as an E3-ubiquitin ligase adaptor for 53BP1

2019 ◽  
Vol 27 (4) ◽  
pp. 1200-1213 ◽  
Author(s):  
Ainhoa Nieto ◽  
Makoto R. Hara ◽  
Victor Quereda ◽  
Wayne Grant ◽  
Vanessa Saunders ◽  
...  
Keyword(s):  
Dna Damage ◽  
Ionizing Radiation ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Molecular Scaffold ◽  
Strand Breaks

Abstract Cellular DNA is constantly under threat from internal and external insults, consequently multiple pathways have evolved to maintain chromosomal fidelity. Our previous studies revealed that chronic stress, mediated by continuous stimulation of the β2-adrenergic-βarrestin-1 signaling axis suppresses activity of the tumor suppressor p53 and impairs genomic integrity. In this pathway, βarrestin-1 (βarr1) acts as a molecular scaffold to promote the binding and degradation of p53 by the E3-ubiquitin ligase, MDM2. We sought to determine whether βarr1 plays additional roles in the repair of DNA damage. Here we demonstrate that in mice βarr1 interacts with p53-binding protein 1 (53BP1) with major consequences for the repair of DNA double-strand breaks. 53BP1 is a principle component of the DNA damage response, and when recruited to the site of double-strand breaks in DNA, 53BP1 plays an important role coordinating repair of these toxic lesions. Here, we report that βarr1 directs 53BP1 degradation by acting as a scaffold for the E3-ubiquitin ligase Rad18. Consequently, knockdown of βarr1 stabilizes 53BP1 augmenting the number of 53BP1 DNA damage repair foci following exposure to ionizing radiation. Accordingly, βarr1 loss leads to a marked increase in irradiation resistance both in cells and in vivo. Thus, βarr1 is an important regulator of double strand break repair, and disruption of the βarr1/53BP1 interaction offers an attractive strategy to protect cells against high levels of exposure to ionizing radiation.

scholarly journals Methyl methanesulfonate (MMS) produces heat-labile DNA damage but no detectable in vivo DNA double-strand breaks

2012 ◽  
Vol 40 (12) ◽  
pp. 5794-5794
Author(s):  
C. Lundin ◽  
M. North ◽  
K. Erixon ◽  
K. Walters ◽  
D. Jenssen ◽  
...  
Keyword(s):  
Dna Damage ◽  
Double Strand Breaks ◽  
Methyl Methanesulfonate ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Heat Labile

scholarly journals DNA double-strand breaks in the Toxoplasma gondii-infected cells by the action of reactive oxygen species

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Haohan Zhuang ◽  
Chaoqun Yao ◽  
Xianfeng Zhao ◽  
Xueqiu Chen ◽  
Yimin Yang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Toxoplasma Gondii ◽  
Double Strand Breaks ◽  
Host Cells ◽  
Oxygen Species ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Damage Response ◽  
Infected Cells

Abstract Background Toxoplasma gondii is an obligate parasite of all warm-blooded animals around the globe. Once infecting a cell, it manipulates the host’s DNA damage response that is yet to be elucidated. The objectives of the present study were three-fold: (i) to assess DNA damages in T. gondii-infected cells in vitro; (ii) to ascertain causes of DNA damage in T. gondii-infected cells; and (iii) to investigate activation of DNA damage responses during T. gondii infection. Methods HeLa, Vero and HEK293 cells were infected with T. gondii at a multiplicity of infection (MOI) of 10:1. Infected cells were analyzed for a biomarker of DNA double-strand breaks (DSBs) γH2AX at 10 h, 20 h or 30 h post-infection using both western blot and immunofluorescence assay. Reactive oxygen species (ROS) levels were measured using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), and ROS-induced DNA damage was inhibited by a ROS inhibitor N-acetylcysteine (NAC). Lastly, DNA damage responses were evaluated by detecting the active form of ataxia telangiectasia mutated/checkpoint kinase 2 (ATM/CHK2) by western blot. Results γH2AX levels in the infected HeLa cells were significantly increased over time during T. gondii infection compared to uninfected cells. NAC treatment greatly reduced ROS and concomitantly diminished γH2AX in host cells. The phosphorylated ATM/CHK2 were elevated in T. gondii-infected cells. Conclusions Toxoplasma gondii infection triggered DNA DSBs with ROS as a major player in host cells in vitro. It also activated DNA damage response pathway ATM/CHK2. Toxoplasma gondii manages to keep a balance between survival and apoptosis of its host cells for the benefit of its own survival.

Heterochromatin and the DNA damage response: the need to relaxThis paper is one of a selection of papers in a Special Issue entitled 31st Annual International Asilomar Chromatin and Chromosomes Conference, and has undergone the Journal’s usual peer review process.

2011 ◽  
Vol 89 (1) ◽  
pp. 45-60 ◽  
Author(s):  
Kendra L. Cann ◽  
Graham Dellaire
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Chromatin Structure ◽  
Peer Review Process ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Atm Kinase ◽  
Strand Breaks ◽  
Damage Response ◽  
Chromosomal Mutations

Higher order chromatin structure has an impact on all nuclear functions, including the DNA damage response. Over the past several years, it has become increasingly clear that heterochromatin and euchromatin represent separate entities with respect to both damage sensitivity and repair. The chromatin compaction present in heterochromatin helps to protect this DNA from damage; however, when lesions do occur, the compaction restricts the ability of DNA damage response proteins to access the site, as evidenced by its ability to block the expansion of H2AX phosphorylation. As such, DNA damage in heterochromatin is refractory to repair, which requires the surrounding chromatin structure to be decondensed. In the case of DNA double-strand breaks, this relaxation is at least partially mediated by the ATM kinase phosphorylating and inhibiting the function of the transcriptional repressor KAP1. This review will focus on the functions of KAP1 and other proteins involved in the maintenance or restriction of heterochromatin, including HP1 and TIP60, in the DNA damage response. As heterochromatin is important for maintaining genomic stability, cells must maintain a delicate balance between allowing repair factors access to these regions and ensuring that these regions retain their organization to prevent increased DNA damage and chromosomal mutations.

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