scholarly journals Distinct pathways of homologous recombination controlled by the SWS1-SWSAP1-SPIDR complex

2020 ◽  
Author(s):  
Rohit Prakash ◽  
Thomas Sandoval ◽  
Florian Morati ◽  
Jennifer A Zagelbaum ◽  
Brett Taylor ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Genetic Interactions ◽  
Repair Pathway ◽  
Embryo Survival ◽  
Poor Growth ◽  
Homology Directed Repair ◽  
Blm Helicase ◽  
Cancer Initiation ◽  
High Level ◽  
Insight Into

AbstractHomology-directed repair (HDR), a critical DNA repair pathway in mammalian cells, is complex, leading to multiple outcomes with different impacts on genomic integrity. However, the factors that control these different outcomes are often not well understood. Here we show that SWS1-SWSAP1-SPIDR controls distinct types of HDR. Despite their requirement for stable assembly of RAD51 recombinase at DNA damage sites, these proteins are not essential for intra-chromosomal HDR, providing insight into why patients and mice with mutations are viable. However, SWS1-SWSAP1-SPIDR is critical for inter-homolog HDR, the first mitotic factor identified specifically for this function. Furthermore, SWS1-SWSAP1-SPIDR drives the high level of sister-chromatid exchange, promotes long-range loss of heterozygosity often involved with cancer initiation, and impels the poor growth of BLM helicase-deficient cells. The relevance of these genetic interactions is evident as SWSAP1 loss prolongs Blm-mutant embryo survival, suggesting a possible druggable target for the treatment of Bloom syndrome.

scholarly journals Distinct pathways of homologous recombination controlled by the SWS1–SWSAP1–SPIDR complex

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Rohit Prakash ◽  
Thomas Sandoval ◽  
Florian Morati ◽  
Jennifer A. Zagelbaum ◽  
Pei-Xin Lim ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Genetic Interactions ◽  
Repair Pathway ◽  
Embryo Survival ◽  
Poor Growth ◽  
Homology Directed Repair ◽  
Blm Helicase ◽  
Cancer Initiation ◽  
High Level ◽  
Insight Into

AbstractHomology-directed repair (HDR), a critical DNA repair pathway in mammalian cells, is complex, leading to multiple outcomes with different impacts on genomic integrity. However, the factors that control these different outcomes are often not well understood. Here we show that SWS1–SWSAP1-SPIDR controls distinct types of HDR. Despite their requirement for stable assembly of RAD51 recombinase at DNA damage sites, these proteins are not essential for intra-chromosomal HDR, providing insight into why patients and mice with mutations are viable. However, SWS1–SWSAP1-SPIDR is critical for inter-homolog HDR, the first mitotic factor identified specifically for this function. Furthermore, SWS1–SWSAP1-SPIDR drives the high level of sister-chromatid exchange, promotes long-range loss of heterozygosity often involved with cancer initiation, and impels the poor growth of BLM helicase-deficient cells. The relevance of these genetic interactions is evident as SWSAP1 loss prolongs Blm-mutant embryo survival, suggesting a possible druggable target for the treatment of Bloom syndrome.

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.

scholarly journals The YadA Protein of Yersinia pseudotuberculosis Mediates High-Efficiency Uptake into Human Cells under Environmental Conditions in Which Invasin Is Repressed

2002 ◽  
Vol 70 (9) ◽  
pp. 4880-4891 ◽  
Author(s):  
Julia Eitel ◽  
Petra Dersch
Keyword(s):  
Extracellular Matrix ◽  
Mammalian Cells ◽  
Yersinia Pseudotuberculosis ◽  
High Efficiency ◽  
Environmental Parameters ◽  
Infection Process ◽  
Nutrient Concentrations ◽  
Integrin Receptors ◽  
Uptake Process ◽  
High Level

ABSTRACT The YadA protein is a major adhesin of Yersinia pseudotuberculosis that promotes tight adhesion to mammalian cells by binding to extracellular matrix proteins. In this study, we first addressed the possibility of competitive interference of YadA and the major invasive factor invasin and found that expression of YadA in the presence of invasin affected neither the export nor the function of invasin in the outer membrane. Furthermore, expression of YadA promoted both bacterial adhesion and high-efficiency invasion entirely independently of invasin. Antibodies against fibronectin and β1 integrins blocked invasion, indicating that invasion occurs via extracellular-matrix-dependent bridging between YadA and the host cell β1 integrin receptors. Inhibitor studies also demonstrated that tyrosine and Ser/Thr kinases, as well as phosphatidylinositol 3-kinase, are involved in the uptake process. Further expression studies revealed that yadA is regulated in response to several environmental parameters, including temperature, ion and nutrient concentrations, and the bacterial growth phase. In complex medium, YadA production was generally repressed but could be induced by addition of Mg2+. Maximal expression of yadA was obtained in exponential-phase cells grown in minimal medium at 37°C, conditions under which the invasin gene is repressed. These results suggest that YadA of Y. pseudotuberculosis constitutes another independent high-level uptake pathway that might complement other cell entry mechanisms (e.g., invasin) at certain sites or stages during the infection process.

scholarly journals Racemization-free synthesis of Nα-2-thiophenoyl-phenylalanine-2-morpholinoanilide enantiomers and their antimycobacterial activity

Amino Acids ◽  
2021 ◽  
Author(s):  
Lea Mann ◽  
Markus Lang ◽  
Philipp Schulze ◽  
Jan Henrik Halz ◽  
René Csuk ◽  
...  
Keyword(s):  
Title Compound ◽  
Mammalian Cells ◽  
Antimycobacterial Activity ◽  
Lead Compound ◽  
Coupling Reagents ◽  
Hydrogen Bonding Interactions ◽  
Amide Coupling ◽  
Subsequent Separation ◽  
Synthetic Routes ◽  
Insight Into

AbstractNα-2-thiophenoyl-d-phenylalanine-2-morpholinoanilide (MMV688845, IUPAC: N-(1-((2-morpholinophenyl)amino)-1-oxo-3-phenylpropan-2-yl)thiophene-2-carboxamide) from the Pathogen Box® library (Medicines for Malaria Ventures, MMV) is a promising lead compound for antimycobacterial drug development. Two straightforward synthetic routes to the title compound starting from phenylalanine or its Boc-protected derivative are reported. Employing Boc-phenylalanine as starting material and the T3P® and PyBOP® amide coupling reagents enables racemization-free synthesis, avoiding the need for subsequent separation of the enantiomers. The crystal structure of the racemic counterpart gives insight into the molecular structure and hydrogen bonding interactions in the solid state. The R-enantiomer of the title compound (derived from d-phenylalanine) exhibits activity against non-pathogenic and pathogenic mycobacterial strains, whereas the S-enantiomer is inactive. Neither of the enantiomers and the racemate of the title compound shows cytotoxicity against various mammalian cells.

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.

Gotta catch ‘em all: the microscale quest to understand cancer biology

2016 ◽  
Vol 8 (12) ◽  
pp. 1203-1207 ◽  
Author(s):  
Zhenwei Ma ◽  
Christopher Moraes
Keyword(s):  
Cancer Biology ◽  
Recent Advances ◽  
Cancer Initiation ◽  
Insight Into

We highlight recent advances in the innovative use of microscale engineered technologies to gain new insight into the integrative biophysical mechanisms that drive cancer initiation and progression.

β-Defensin overexpression induces progressive muscle degeneration in mice

2007 ◽  
Vol 292 (6) ◽  
pp. C2141-C2149 ◽  
Author(s):  
Yasuhiro Yamaguchi ◽  
Takahide Nagase ◽  
Tetsuji Tomita ◽  
Kyoko Nakamura ◽  
Shigetomo Fukuhara ◽  
...  
Keyword(s):  
Muscular Dystrophy ◽  
Transgenic Mice ◽  
Mammalian Cells ◽  
Tissue Injury ◽  
Elevated Serum ◽  
Creatine Kinase Activity ◽  
Poor Growth ◽  
Short Lifespan ◽  
Host Defense System ◽  
Muscle Impairment

Defensins comprise a family of cationic antimicrobial peptides characterized by conserved cysteine residues. They are produced in various organs including skeletal muscle and are identified as key elements in the host defense system as potent effectors. At the same time, defensins have potential roles in the regulation of inflammation and, furthermore, can exert cytotoxic effects on several mammalian cells. Here, we developed transgenic mice overexpressing mouse β-defensin-6 to explore the pathophysiological roles of the defensin family as a novel mediator of inflammatory tissue injury. Unexpectedly, the transgenic mice showed short lifespan, poor growth, and progressive myofiber degeneration with functional muscle impairment, predominant centronucleated myofibers, and elevated serum creatine kinase activity, as seen in human muscular dystrophy. Furthermore, some of the transgenic myofibers showed IκBα accumulation, which would be related to the myofiber apoptosis of limb-girdle muscular dystrophy type 2A. The present findings may unravel a concealed linkage between the innate immune system and the pathophysiology of degenerative diseases.

Mutations Causing Constitutive Invertase Synthesis in Yeast: Genetic Interactions with snf Mutations

Genetics ◽  
1987 ◽  
Vol 115 (2) ◽  
pp. 247-253
Author(s):  
Lenore Neigeborn ◽  
Marian Carlson
Keyword(s):  
Regulatory Region ◽  
Constitutive Expression ◽  
Genetic Interactions ◽  
The Third ◽  
Recessive Mutations ◽  
Constitutive Mutation ◽  
Complementation Groups ◽  
High Level ◽  
Yeast Genetic ◽  
Very High

ABSTRACT We have selected 210 mutants able to grow on sucrose in the presence of 2-deoxyglucose. We identified recessive mutations in three major complementation groups that cause constitutive (glucose-insensitive) secreted invertase synthesis. Two groups comprise alleles of the previously identified HXK2 and REG1 genes, and the third group was designated cid1 (constitutive invertase derepression). The effect of cid1 on SUC2 expression is mediated by the SUC2 upstream regulatory region, as judged by the constitutive expression of a SUC2-LEU2-lacZ fusion in which the LEU2 promoter is under control of SUC2 upstream sequences. A cid1 mutation also causes glucose-insensitive expression of maltase. The previously isolated constitutive mutation ssn6 is epistatic to cid1, reg1 and hxk2 for very high level constitutive invertase expression. Mutations in SNF genes that prevent derepression of invertase are epistatic to cid1, reg1 and hxk2; we have previously shown that ssn6 has different epistasis relationships with snf mutations. The constitutive mutation tup1 was found to resemble ssn6 in its genetic interactions with snf mutations. These findings suggest that CID1, REG1 and HXK2 are functionally distinct from SSN6 and TUP1.

Insight into the formation of a continuous sheath structure for the PS phase in tri-continuous PVDF/PS/HDPE blends

RSC Advances ◽  
2016 ◽  
Vol 6 (1) ◽  
pp. 439-447 ◽  
Author(s):  
Rui Dou ◽  
Shuanglin Li ◽  
Yan Shao ◽  
Bo Yin ◽  
Mingbo Yang
Keyword(s):  
Ternary Blends ◽  
Continuous Structure ◽  
High Level ◽  
Very High ◽  
Insight Into ◽  
Sheath Structure

A hierarchical tri-continuous structure is formed and controlled in PVDF/PS/HDPE ternary blends. A very high level of PS continuity, about 80%, is achieved only with a PS volume composition as low as 11 vol%.

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