scholarly journals The Saccharomyces cerevisiae RAD6 Group Is Composed of an Error-Prone and Two Error-Free Postreplication Repair Pathways

Genetics ◽  
2000 ◽  
Vol 155 (4) ◽  
pp. 1633-1641 ◽  
Author(s):  
Wei Xiao ◽  
Barbara L Chow ◽  
Stacey Broomfield ◽  
Michelle Hanna
Keyword(s):  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Signal Transducer ◽  
Repair Pathway ◽  
Polyubiquitin Chain ◽  
Postreplication Repair ◽  
Translesion Dna Synthesis ◽  
Radiation Repair ◽  
Repair Pathways ◽  
High Degree

Abstract The RAD6 postreplication repair and mutagenesis pathway is the only major radiation repair pathway yet to be extensively characterized. It has been previously speculated that the RAD6 pathway consists of two parallel subpathways, one error free and another error prone (mutagenic). Here we show that the RAD6 group genes can be exclusively divided into three rather than two independent subpathways represented by the RAD5, POL30, and REV3 genes; the REV3 pathway is largely mutagenic, whereas the RAD5 and the POL30 pathways are deemed error free. Mutants carrying characteristic mutations in each of the three subpathways are phenotypically indistinguishable from a single mutant such as rad18, which is defective in the entire RAD6 postreplication repair/tolerance pathway. Furthermore, the rad18 mutation is epistatic to all single or combined mutations in any of the above three subpathways. Our data also suggest that MMS2 and UBC13 play a key role in coordinating the response of the error-free subpathways; Mms2 and Ubc13 form a complex required for a novel polyubiquitin chain assembly, which probably serves as a signal transducer to promote both RAD5 and POL30 error-free postreplication repair pathways. The model established by this study will facilitate further research into the molecular mechanisms of postreplication repair and translesion DNA synthesis. In view of the high degree of sequence conservation of the RAD6 pathway genes among all eukaryotes, the model presented in this study may also apply to mammalian cells and predicts links to human diseases.

scholarly journals A Targeted and Tuneable DNA Damage Tool Using CRISPR/Cas9

Biomolecules ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 288
Author(s):  
Ioannis Emmanouilidis ◽  
Natalia Fili ◽  
Alexander W. Cook ◽  
Yukti Hari-Gupta ◽  
Ália dos Santos ◽  
...  
Keyword(s):  
Dna Damage ◽  
Mammalian Cells ◽  
Genome Stability ◽  
Molecular Mechanisms ◽  
Low Cost ◽  
Strand Breaks ◽  
Guide Rnas ◽  
Area Of Interest ◽  
Culture Models ◽  
Repair Pathways

Mammalian cells are constantly subjected to a variety of DNA damaging events that lead to the activation of DNA repair pathways. Understanding the molecular mechanisms of the DNA damage response allows the development of therapeutics which target elements of these pathways. Double-strand breaks (DSB) are particularly deleterious to cell viability and genome stability. Typically, DSB repair is studied using DNA damaging agents such as ionising irradiation or genotoxic drugs. These induce random lesions at non-predictive genome sites, where damage dosage is difficult to control. Such interventions are unsuitable for studying how different DNA damage recognition and repair pathways are invoked at specific DSB sites in relation to the local chromatin state. The RNA-guided Cas9 (CRISPR-associated protein 9) endonuclease enzyme is a powerful tool to mediate targeted genome alterations. Cas9-based genomic intervention is attained through DSB formation in the genomic area of interest. Here, we have harnessed the power to induce DSBs at defined quantities and locations across the human genome, using custom-designed promiscuous guide RNAs, based on in silico predictions. This was achieved using electroporation of recombinant Cas9-guide complex, which provides a generic, low-cost and rapid methodology for inducing controlled DNA damage in cell culture models.

scholarly journals A targeted and tuneable DNA damage tool using CRISPR/Cas9

2021 ◽  
Author(s):  
Ioannis Emmanouilidis ◽  
Natalia Fili ◽  
Alexander W. Cook ◽  
Yukti Hari-Gupta ◽  
Ália dos Santos ◽  
...  
Keyword(s):  
Dna Damage ◽  
Mammalian Cells ◽  
Genome Stability ◽  
Molecular Mechanisms ◽  
Low Cost ◽  
Strand Breaks ◽  
Guide Rnas ◽  
Area Of Interest ◽  
Culture Models ◽  
Repair Pathways

ABSTRACTMammalian cells are constantly subjected to a variety of DNA damaging events that lead to the activation of DNA repair pathways. Understanding the molecular mechanisms of the DNA damage response allows the development of therapeutics which target elements of these pathways.Double-Strand Breaks (DSB) are particularly deleterious to cell viability and genome stability. Typically, DSB repair is studied using DNA damaging agents such as ionising irradiation or genotoxic drugs. These induce random lesions at non-predictive genome sites, where damage dosage is difficult to control. Such interventions are unsuitable for studying how different DNA damage recognition and repair pathways are invoked at specific DSB sites in relation to the local chromatin state.The RNA-guided Cas9 (CRISPR associated protein 9) endonuclease enzyme, is a powerful tool to mediate targeted genome alterations. Cas9-based genomic intervention is attained through DSB formation in the genomic area of interest. Here, we have harnessed the power to induce DSBs at defined quantities and locations across the human genome, using custom-designed promiscuous guide RNAs, based on in silico predictions. This was achieved using electroporation of recombinant Cas9-guide complex which provides a generic, low-cost and rapid methodology for inducing controlled DNA damage in cell culture models.

scholarly journals Methods Favoring Homology-Directed Repair Choice in Response to CRISPR/Cas9 Induced-Double Strand Breaks

2020 ◽  
Vol 21 (18) ◽  
pp. 6461
Author(s):  
Han Yang ◽  
Shuling Ren ◽  
Siyuan Yu ◽  
Haifeng Pan ◽  
Tingdong Li ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Gene Editing ◽  
Initial Step ◽  
Double Strand Breaks ◽  
Repair Pathway ◽  
End Joining ◽  
Strand Breaks ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Repair Pathways

Precise gene editing is—or will soon be—in clinical use for several diseases, and more applications are under development. The programmable nuclease Cas9, directed by a single-guide RNA (sgRNA), can introduce double-strand breaks (DSBs) in target sites of genomic DNA, which constitutes the initial step of gene editing using this novel technology. In mammals, two pathways dominate the repair of the DSBs—nonhomologous end joining (NHEJ) and homology-directed repair (HDR)—and the outcome of gene editing mainly depends on the choice between these two repair pathways. Although HDR is attractive for its high fidelity, the choice of repair pathway is biased in a biological context. Mammalian cells preferentially employ NHEJ over HDR through several mechanisms: NHEJ is active throughout the cell cycle, whereas HDR is restricted to S/G2 phases; NHEJ is faster than HDR; and NHEJ suppresses the HDR process. This suggests that definitive control of outcome of the programmed DNA lesioning could be achieved through manipulating the choice of cellular repair pathway. In this review, we summarize the DSB repair pathways, the mechanisms involved in choice selection based on DNA resection, and make progress in the research investigating strategies that favor Cas9-mediated HDR based on the manipulation of repair pathway choice to increase the frequency of HDR in mammalian cells. The remaining problems in improving HDR efficiency are also discussed. This review should facilitate the development of CRISPR/Cas9 technology to achieve more precise gene editing.

Dissection of the molecular mechanisms of protein targeting to the peroxisome using immunofluorescence and immunocryoelectron microscopies

1990 ◽  
Vol 48 (3) ◽  
pp. 44-45
Author(s):  
G-A. Keller ◽  
S. J. Gould ◽  
S. Subramani ◽  
S. Krisans
Keyword(s):  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Protein Targeting ◽  
Firefly Luciferase ◽  
Peroxisomal Enzyme ◽  
Transfected Cells ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Series Of Experiments ◽  
Peroxisomal Targeting Signal

Subcellular compartments within eukaryotic cells must each be supplied with unique sets of proteins that must be directed to, and translocated across one or more membranes of the target organelles. This transport is mediated by cis- acting targeting signals present within the imported proteins. The following is a chronological account of a series of experiments designed and carried out in an effort to understand how proteins are targeted to the peroxisomal compartment.-We demonstrated by immunocryoelectron microscopy that the enzyme luciferase is a peroxisomal enzyme in the firefly lantern. -We expressed the cDNA encoding firefly luciferase in mammalian cells and demonstrated by immunofluorescence that the enzyme was transported into the peroxisomes of the transfected cells. -Using deletions, linker insertions, and gene fusion to identify regions of luciferase involved in its transport to the peroxisomes, we demonstrated that luciferase contains a peroxisomal targeting signal (PTS) within its COOH-terminal twelve amino acid.

A Possible Modulation Mechanism of Intramolecular and Intermolecular Interactions for NCAM Polysialylation and Cell Migration

2019 ◽  
Vol 19 (25) ◽  
pp. 2271-2282 ◽  
Author(s):  
Bo Lu ◽  
Xue-Hui Liu ◽  
Si-Ming Liao ◽  
Zhi-Long Lu ◽  
Dong Chen ◽  
...  
Keyword(s):  
Cell Migration ◽  
Intermolecular Interactions ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Intramolecular Interaction ◽  
Polysialic Acid ◽  
Tumor Cell Migration ◽  
Neural Cell Adhesion ◽  
Polybasic Domains ◽  
Novel Model

Polysialic acid (polySia) is a novel glycan that posttranslationally modifies neural cell adhesion molecules (NCAMs) in mammalian cells. Up-regulation of polySia-NCAM expression or NCAM polysialylation is associated with tumor cell migration and progression in many metastatic cancers and neurocognition. It has been known that two highly homologous mammalian polysialyltransferases (polySTs), ST8Sia II (STX) and ST8Sia IV (PST), can catalyze polysialylation of NCAM, and two polybasic domains, polybasic region (PBR) and polysialyltransferase domain (PSTD) in polySTs play key roles in affecting polyST activity or NCAM polysialylation. However, the molecular mechanisms of NCAM polysialylation and cell migration are still not entirely clear. In this minireview, the recent research results about the intermolecular interactions between the PBR and NCAM, the PSTD and cytidine monophosphate-sialic acid (CMP-Sia), the PSTD and polySia, and as well as the intramolecular interaction between the PBR and the PSTD within the polyST, are summarized. Based on these cooperative interactions, we have built a novel model of NCAM polysialylation and cell migration mechanisms, which may be helpful to design and develop new polysialyltransferase inhibitors.

scholarly journals Control of PKR Protein Kinase by Hepatitis C Virus Nonstructural 5A Protein: Molecular Mechanisms of Kinase Regulation

1998 ◽  
Vol 18 (9) ◽  
pp. 5208-5218 ◽  
Author(s):  
Michael Gale ◽  
Collin M. Blakely ◽  
Bart Kwieciszewski ◽  
Seng-Lai Tan ◽  
Michelle Dossett ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protein Kinase ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Functional Assay ◽  
Binding Region ◽  
Dimerization Domain ◽  
Antiviral Effects

ABSTRACT The PKR protein kinase is a critical component of the cellular antiviral and antiproliferative responses induced by interferons. Recent evidence indicates that the nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) can repress PKR function in vivo, possibly allowing HCV to escape the antiviral effects of interferon. NS5A presents a unique tool by which to study the molecular mechanisms of PKR regulation in that mutations within a region of NS5A, termed the interferon sensitivity-determining region (ISDR), are associated with sensitivity of HCV to the antiviral effects of interferon. In this study, we investigated the mechanisms of NS5A-mediated PKR regulation and the effect of ISDR mutations on this regulatory process. We observed that the NS5A ISDR, though necessary, was not sufficient for PKR interactions; we found that an additional 26 amino acids (aa) carboxyl to the ISDR were required for NS5A-PKR complex formation. Conversely, we localized NS5A binding to within PKR aa 244 to 296, recently recognized as a PKR dimerization domain. Consistent with this observation, we found that NS5A from interferon-resistant HCV genotype 1b disrupted kinase dimerization in vivo. NS5A-mediated disruption of PKR dimerization resulted in repression of PKR function and inhibition of PKR-mediated eIF-2α phosphorylation. Introduction of multiple ISDR mutations abrogated the ability of NS5A to bind to PKR in mammalian cells and to inhibit PKR in a yeast functional assay. These results indicate that mutations within the PKR-binding region of NS5A, including those within the ISDR, can disrupt the NS5A-PKR interaction, possibly rendering HCV sensitive to the antiviral effects of interferon. We propose a model of PKR regulation by NS5A which may have implications for therapeutic strategies against HCV.

scholarly journals Molecular analysis of ethyl methanesulfonate-induced reversion of a chromosomally integrated mutant shuttle vector gene in mammalian cells.

1988 ◽  
Vol 8 (10) ◽  
pp. 4185-4189 ◽  
Author(s):  
J A Greenspan ◽  
F M Xu ◽  
R L Davidson
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Shuttle Vector ◽  
Ethyl Methanesulfonate ◽  
Wild Type ◽  
Single Base ◽  
Type Sequence

The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC----AT transitions or AT----TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT----GC transitions and for one cell line with a GC----CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT----GC transitions had mutated back to the wild-type gpt sequence via GC----AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC----CG transversion had mutated via GC----AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG----GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.

scholarly journals Signal Transducer and Activator of Transcription 3 (STAT3) Suppresses STAT1/Interferon Signaling Pathway and Inflammation in Senescent Preadipocytes

Antioxidants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 334
Author(s):  
Aisha Y. Madani ◽  
Yasser Majeed ◽  
Houari B. Abdesselem ◽  
Maha V. Agha ◽  
Muneera Vakayil ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Molecular Mechanisms ◽  
Adenosine Monophosphate ◽  
Human Adipose Tissue ◽  
Premature Aging ◽  
Guanosine Monophosphate ◽  
Negative Regulator ◽  
Signal Transducer ◽  
Interferon Signaling

Obesity promotes premature aging and dysfunction of white adipose tissue (WAT) through the accumulation of cellular senescence. The senescent cells burden in WAT has been linked to inflammation, insulin-resistance (IR), and type 2 diabetes (T2D). There is limited knowledge about molecular mechanisms that sustain inflammation in obese states. Here, we describe a robust and physiologically relevant in vitro system to trigger senescence in mouse 3T3-L1 preadipocytes. By employing transcriptomics analyses, we discovered up-regulation of key pro-inflammatory molecules and activation of interferon/signal transducer and activator of transcription (STAT)1/3 signaling in senescent preadipocytes, and expression of downstream targets was induced in epididymal WAT of obese mice, and obese human adipose tissue. To test the relevance of STAT1/3 signaling to preadipocyte senescence, we used Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9) technology to delete STAT1/3 and discovered that STAT1 promoted growth arrest and cooperated with cyclic Guanosine Monophosphate-Adenosine Monophosphate (GMP-AMP) synthase-stimulator of interferon genes (cGAS-STING) to drive the expression of interferon β (IFNβ), C-X-C motif chemokine ligand 10 (CXCL10), and interferon signaling-related genes. In contrast, we discovered that STAT3 was a negative regulator of STAT1/cGAS-STING signaling—it suppressed senescence and inflammation. These data provide insights into how STAT1/STAT3 signaling coordinates senescence and inflammation through functional interactions with the cGAS/STING pathway.

scholarly journals Phosphorylation: The Molecular Switch of Double-Strand Break Repair

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
K. C. Summers ◽  
F. Shen ◽  
E. A. Sierra Potchanant ◽  
E. A. Phipps ◽  
R. J. Hickey ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Genomic Stability ◽  
Molecular Switches ◽  
Strand Break ◽  
Nonhomologous End Joining ◽  
End Joining ◽  
Brca1 And Brca2 ◽  
Damage Response ◽  
Repair Mechanisms ◽  
Repair Pathways

Repair of double-stranded breaks (DSBs) is vital to maintaining genomic stability. In mammalian cells, DSBs are resolved in one of the following complex repair pathways: nonhomologous end-joining (NHEJ), homologous recombination (HR), or the inclusive DNA damage response (DDR). These repair pathways rely on factors that utilize reversible phosphorylation of proteins as molecular switches to regulate DNA repair. Many of these molecular switches overlap and play key roles in multiple pathways. For example, the NHEJ pathway and the DDR both utilize DNA-PK phosphorylation, whereas the HR pathway mediates repair with phosphorylation of RPA2, BRCA1, and BRCA2. Also, the DDR pathway utilizes the kinases ATM and ATR, as well as the phosphorylation of H2AX and MDC1. Together, these molecular switches regulate repair of DSBs by aiding in DSB recognition, pathway initiation, recruitment of repair factors, and the maintenance of repair mechanisms.

scholarly journals Molecular Mechanisms of the Whole DNA Repair System: A Comparison of Bacterial and Eukaryotic Systems

2010 ◽  
Vol 2010 ◽  
pp. 1-32 ◽  
Author(s):  
Rihito Morita ◽  
Shuhei Nakane ◽  
Atsuhiro Shimada ◽  
Masao Inoue ◽  
Hitoshi Iino ◽  
...  
Keyword(s):  
Dna Repair ◽  
Molecular Mechanisms ◽  
Excision Repair ◽  
Thermus Thermophilus ◽  
Repair System ◽  
System A ◽  
Recombination Repair ◽  
Repair Mechanisms ◽  
Repair Pathways ◽  
Base Excision

DNA is subjected to many endogenous and exogenous damages. All organisms have developed a complex network of DNA repair mechanisms. A variety of different DNA repair pathways have been reported: direct reversal, base excision repair, nucleotide excision repair, mismatch repair, and recombination repair pathways. Recent studies of the fundamental mechanisms for DNA repair processes have revealed a complexity beyond that initially expected, with inter- and intrapathway complementation as well as functional interactions between proteins involved in repair pathways. In this paper we give a broad overview of the whole DNA repair system and focus on the molecular basis of the repair machineries, particularly inThermus thermophilusHB8.

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