scholarly journals Virulence and Karyotype Analyses of rad52Mutants of Candida albicans: Regeneration of a TruncatedChromosome of a Reintegrant Strain (rad52/RAD52) in the Host

2005 ◽  
Vol 73 (12) ◽  
pp. 8069-8078 ◽  
Author(s):  
Neeraj Chauhan ◽  
Toni Ciudad ◽  
Ane Rodríguez-Alejandre ◽  
Germán Larriba ◽  
Richard Calderone ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Large Deletion ◽  
Nonhomologous End Joining ◽  
Pcr Analysis ◽  
Wild Type ◽  
End Joining ◽  
Inoculum Concentration ◽  
A Cell ◽  
Karyotype Analyses ◽  
Cell Inoculum

ABSTRACT The virulence of Candida albicans mutants lacking one or both copies of RAD52, a gene involved in homologous recombination (HR), was evaluated in a murine model of hematogenously disseminated candidiasis. In this study, the virulence of the rad52Δ mutant was dependent upon the inoculum concentration. Mice survived at a cell inoculum of 1 × 106, but there was a decrease in survival time at dosages of 1.5 × 106 and especially at 3 × 106 cells per animal. The heterozygote RAD52/rad52 behaved like wild type, whereas a reintegrant strain was intermediate in its ability to cause death compared to these strains and to the avirulent rad52/rad52 null at inocula of 1 × 106 and 1.5 × 106 cells. A double mutant, lig4/lig4/rad52/rad52, was avirulent at all inocula used. PCR analysis of the RAD52 and/or LIG4 loci showed that all strains recovered from animals matched the genotype of the inoculated strains. Analysis of the electrophoretical karyotypes indicated that the inoculated, reintegrant strain carried a large deletion in one copy of chromosome 6 (the shortest homologue, or Chr6b). Interestingly, truncated Chr6b was regenerated in all the strains recovered from moribund animals using the homologue as a template. Further, regeneration of Chr6b was paralleled by an increase in virulence that was still lower than that of wild type, likely because of the persistent loss of heterozygosity in the regenerated region. Overall, our results indicate that systemic candidiasis can develop in the absence of HR, but simultaneous elimination of both recombination pathways, HR and nonhomologous end-joining, suppresses virulence even at very high inocula.

scholarly journals Phenotypic Analysis and Virulence of Candida albicans LIG4 Mutants

2001 ◽  
Vol 69 (1) ◽  
pp. 137-147 ◽  
Author(s):  
Encarnación Andaluz ◽  
Richard Calderone ◽  
Guadalupe Reyes ◽  
Germán Larriba
Keyword(s):  
Candida Albicans ◽  
Uv Light ◽  
Significant Rise ◽  
Nonhomologous End Joining ◽  
Mrna Levels ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Phenotypic Analysis ◽  
Positive Role ◽  
Gene Deletions

ABSTRACT In previous studies, we reported the isolation and preliminary characterization of a DNA ligase-encoding gene of Candida albicans. This gene (LIG4) is the structural and functional homologue of both yeast and human ligase IV, which is involved in nonhomologous end joining (NHEJ) of DNA double-strand breaks. In the present study, we have shown that there are no otherLIG4 homologues in C. albicans. In order to study the function of LIG4 in morphogenesis and virulence, we constructed gene deletions. LIG4 transcript levels were reduced in the heterozygote and were completely absent in null strains. Concomitantly, the heterozygote showed a pronounced defect in myceliation, which was slightly greater in the null strain. This was true with several solid and liquid media, such as Spider medium, medium 199, and 2% glucose–1% yeast extract–2% Bacto Peptone, at several pHs. Reintroduction of the wild-type allele into the null mutant partially restored the ability of cells to form hyphae. In agreement with the positive role of LIG4 in morphogenesis, we detected a significant rise in mRNA levels during the morphological transition. LIG4 is not essential for DNA replication or for the repair of DNA damage induced by ionizing radiation or UV light, indicating that these lesions are repaired primarily by homologous recombination. However, our data show that the NHEJ apparatus ofC. albicans may control morphogenesis in this diploid organism. In addition, deletion of one or both copies ofLIG4 resulted in attenuation of virulence in a murine model of candidiasis.

scholarly journals Role of DNA Repair by Nonhomologous-End Joining in Bacillus subtilis Spore Resistance to Extreme Dryness, Mono- and Polychromatic UV, and Ionizing Radiation

2007 ◽  
Vol 189 (8) ◽  
pp. 3306-3311 ◽  
Author(s):  
Ralf Moeller ◽  
Erko Stackebrandt ◽  
Günther Reitz ◽  
Thomas Berger ◽  
Petra Rettberg ◽  
...  
Keyword(s):  
Dna Repair ◽  
Bacillus Subtilis ◽  
Ionizing Radiation ◽  
Ultrahigh Vacuum ◽  
Nonhomologous End Joining ◽  
Wild Type ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Spore Resistance

ABSTRACT The role of DNA repair by nonhomologous-end joining (NHEJ) in spore resistance to UV, ionizing radiation, and ultrahigh vacuum was studied in wild-type and DNA repair mutants (recA, splB, ykoU, ykoV, and ykoU ykoV mutants) of Bacillus subtilis. NHEJ-defective spores with mutations in ykoU, ykoV, and ykoU ykoV were significantly more sensitive to UV, ionizing radiation, and ultrahigh vacuum than wild-type spores, indicating that NHEJ provides an important pathway during spore germination for repair of DNA double-strand breaks.

scholarly journals Pervasive head-to-tail insertions of DNA templates mask desired CRISPR-Cas9–mediated genome editing events

2020 ◽  
Vol 6 (7) ◽  
pp. eaax2941 ◽  
Author(s):  
Boris V. Skryabin ◽  
Delf-Magnus Kummerfeld ◽  
Leonid Gubar ◽  
Birte Seeger ◽  
Helena Kaiser ◽  
...  
Keyword(s):  
Conditional Knockout ◽  
Nonhomologous End Joining ◽  
High Rate ◽  
Model Organisms ◽  
Pcr Analysis ◽  
End Joining ◽  
Dna Templates ◽  
Homology Directed Repair ◽  
Conditional Knockout Mouse ◽  
Wide Range

CRISPR-Cas9–mediated homology-directed DNA repair is the method of choice for precise gene editing in a wide range of model organisms, including mouse and human. Broad use by the biomedical community refined the method, making it more efficient and sequence specific. Nevertheless, the rapidly evolving technique still contains pitfalls. During the generation of six different conditional knockout mouse models, we discovered that frequently (sometimes solely) homology-directed repair and/or nonhomologous end joining mechanisms caused multiple unwanted head-to-tail insertions of donor DNA templates. Disturbingly, conventionally applied PCR analysis, in most cases, failed to identify these multiple integration events, which led to a high rate of falsely claimed precisely edited alleles. We caution that comprehensive analysis of modified alleles is essential and offer practical solutions to correctly identify precisely edited chromosomes.

scholarly journals Pyridines and Pyrimidines Mediating Activity against an Efflux-Negative Strain of Candida albicans through Putative Inhibition of Lanosterol Demethylase

2004 ◽  
Vol 48 (1) ◽  
pp. 313-318 ◽  
Author(s):  
Ed T. Buurman ◽  
April E. Blodgett ◽  
Kenneth G. Hull ◽  
Daniel Carcanague
Keyword(s):  
Candida Albicans ◽  
Antifungal Activity ◽  
Ergosterol Biosynthesis ◽  
Clear Correlation ◽  
Wild Type ◽  
Inhibition Of Growth ◽  
Ergosterol Synthesis ◽  
A Cell ◽  
Sterol Profile ◽  
Lanosterol Demethylase

ABSTRACT The first step in ergosterol biosynthesis in Saccharomyces cerevisiae consists of the condensation of two acetyl coenzyme A (acetyl-CoA) moieties by acetoacetyl-CoA thiolase, encoded by ERG10. The inhibition of the sterol pathway results in feedback activation of ERG10 transcription. A cell-based reporter assay, in which increased ERG10 transcription results in elevated specific β-galactosidase activity, was used to find novel inhibitors of ergosterol biosynthesis that could serve as chemical starting points for the development of novel antifungal agents. A class of pyridines and pyrimidines identified in this way had no detectable activity against the major fungal pathogen Candida albicans (MICs > 64 μg · ml−1). However, a strain of C. albicans lacking the Cdr1p and Cdr2p efflux pumps was sensitive to the compounds (with MICs ranging from 2 to 64 μg · ml−1), suggesting that they are efficiently removed from wild-type cells. Quantitative analysis of sterol intermediates that accumulated during growth inhibition revealed the accumulation of lanosterol at the expense of ergosterol. Furthermore, a clear correlation was found between the 50% inhibitory concentration at which the sterol profile was altered and the antifungal activity, measured as the MIC. This finding strongly suggests that the inhibition of growth was caused by a reduction in ergosterol synthesis. The compounds described here are a novel class of antifungal pyridines and pyrimidines and the first pyri(mi)dines to be shown to putatively mediate their antifungal activity against C. albicans via lanosterol demethylase.

scholarly journals Nonhomologous end-joining of site-specific but not of radiation-induced DNA double-strand breaks is reduced in the presence of wild-type p53

Oncogene ◽  
2005 ◽  
Vol 24 (10) ◽  
pp. 1663-1672 ◽  
Author(s):  
Jochen Dahm-Daphi ◽  
Petra Hubbe ◽  
Fruzsina Horvath ◽  
Raafat A El-Awady ◽  
Katie E Bouffard ◽  
...  
Keyword(s):  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Wild Type ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Site Specific ◽  
Strand Breaks ◽  
Radiation Induced ◽  
Wild Type P53

JAK2 stimulates homologous recombination and genetic instability: potential implication in the heterogeneity of myeloproliferative disorders

Blood ◽  
2008 ◽  
Vol 112 (4) ◽  
pp. 1402-1412 ◽  
Author(s):  
Isabelle Plo ◽  
Mayuka Nakatake ◽  
Laurent Malivert ◽  
Jean-Pierre de Villartay ◽  
Stéphane Giraudier ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Genetic Instability ◽  
Fold Increase ◽  
Myeloproliferative Disorders ◽  
Nonhomologous End Joining ◽  
Single Mutation ◽  
Wild Type ◽  
End Joining ◽  
Potential Implication ◽  
Receptor Mutant

Abstract The JAK2V617F mutation is frequently observed in classical myeloproliferative disorders, and disease progression is associated with a biallelic acquisition of the mutation occurring by mitotic recombination. In this study, we examined whether JAK2 activation could lead to increased homologous recombination (HR) and genetic instability. In a Ba/F3 cell line expressing the erythropoietin (EPO) receptor, mutant JAK2V617F and, to a lesser extent, wild-type (wt) JAK2 induced an increase in HR activity in the presence of EPO without modifying nonhomologous end-joining efficiency. Moreover, a marked augmentation in HR activity was found in CD34+-derived cells isolated from patients with polycythemia vera or primitive myelofibrosis compared with control samples. This increase was associated with a spontaneous RAD51 foci formation. As a result, sister chromatid exchange was 50% augmented in JAK2V617F Ba/F3 cells compared with JAK2wt cells. Moreover, JAK2 activation increased centrosome and ploidy abnormalities. Finally, in JAK2V617F Ba/F3 cells, we found a 100-fold and 10-fold increase in mutagenesis at the HPRT and Na/K ATPase loci, respectively. Together, this work highlights a new molecular mechanism for HR regulation mediated by JAK2 and more efficiently by JAK2V617F. Our study might provide some keys to understand how a single mutation can give rise to different pathologies.

Nonhomologous end-joining in a cell-free extract from the cultured silkworm cell line BmN4

2005 ◽  
Vol 32 (1) ◽  
pp. 25-34 ◽  
Author(s):  
Arisa Ohsaki ◽  
Kazuhiro Iiyama ◽  
Yoshitaka Miyagawa ◽  
Yutaka Kawaguchi ◽  
Katsumi Koga ◽  
...  
Keyword(s):  
Cell Line ◽  
Nonhomologous End Joining ◽  
Cell Free Extract ◽  
End Joining ◽  
A Cell

scholarly journals Nej1p, a cell type-specific regulator of nonhomologous end joining in yeast

2001 ◽  
Vol 11 (20) ◽  
pp. 1611-1617 ◽  
Author(s):  
Andreas Kegel ◽  
Jimmy O.O. Sjöstrand ◽  
Stefan U. Åström
Keyword(s):  
Nonhomologous End Joining ◽  
Cell Type ◽  
End Joining ◽  
A Cell ◽  
Cell Type Specific

Reciprocal Translocations in Saccharomyces cerevisiae Formed by Nonhomologous End Joining

Genetics ◽  
2004 ◽  
Vol 166 (2) ◽  
pp. 741-751 ◽  
Author(s):  
Xin Yu ◽  
Abram Gabriel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosomal Rearrangements ◽  
Double Strand Breaks ◽  
Open Reading Frames ◽  
Nonhomologous End Joining ◽  
Wild Type ◽  
End Joining ◽  
Reciprocal Translocations ◽  
Strand Breaks ◽  
Reading Frames

Abstract Reciprocal translocations are common in cancer cells, but their creation is poorly understood. We have developed an assay system in Saccharomyces cerevisiae to study reciprocal translocation formation in the absence of homology. We induce two specific double-strand breaks (DSBs) simultaneously on separate chromosomes with HO endonuclease and analyze the subsequent chromosomal rearrangements among surviving cells. Under these conditions, reciprocal translocations via nonhomologous end joining (NHEJ) occur at frequencies of ∼2-7 × 10-5/cell exposed to the DSBs. Yku80p is a component of the cell’s NHEJ machinery. In its absence, reciprocal translocations still occur, but the junctions are associated with deletions and extended overlapping sequences. After induction of a single DSB, translocations and inversions are recovered in wild-type and rad52 strains. In these rearrangements, a nonrandom assortment of sites have fused to the DSB, and their junctions show typical signs of NHEJ. The sites tend to be between open reading frames or within Ty1 LTRs. In some cases the translocation partner is formed by a break at a cryptic HO recognition site. Our results demonstrate that NHEJ-mediated reciprocal translocations can form in S. cerevisiae as a consequence of DSB repair.

scholarly journals ATP-Dependent Ligases and AEP Primases Affect the Profile and Frequency of Mutations in Mycobacteria under Oxidative Stress

Genes ◽  
2021 ◽  
Vol 12 (4) ◽  
pp. 547
Author(s):  
Anna Brzostek ◽  
Filip Gąsior ◽  
Jakub Lach ◽  
Lidia Żukowska ◽  
Ewelina Lechowicz ◽  
...  
Keyword(s):  
Wild Type Strain ◽  
Excision Repair ◽  
Strand Break ◽  
Nonhomologous End Joining ◽  
The Other ◽  
Wild Type ◽  
End Joining ◽  
Protein Mutants ◽  
Ku Protein ◽  
Base Excision

The mycobacterial nonhomologous end-joining pathway (NHEJ) involved in double-strand break (DSB) repair consists of the multifunctional ATP-dependent ligase LigD and the DNA bridging protein Ku. The other ATP-dependent ligases LigC and AEP-primase PrimC are considered as backup in this process. The engagement of LigD, LigC, and PrimC in the base excision repair (BER) process in mycobacteria has also been postulated. Here, we evaluated the sensitivity of Mycolicibacterium smegmatis mutants defective in the synthesis of Ku, Ku-LigD, and LigC1-LigC2-PrimC, as well as mutants deprived of all these proteins to oxidative and nitrosative stresses, with the most prominent effect observed in mutants defective in the synthesis of Ku protein. Mutants defective in the synthesis of LigD or PrimC/LigC presented a lower frequency of spontaneous mutations than the wild-type strain or the strain defective in the synthesis of Ku protein. As identified by whole-genome sequencing, the most frequent substitutions in all investigated strains were T→G and A→C. Double substitutions, as well as insertions of T or CG, were exclusively identified in the strains carrying functional Ku and LigD proteins. On the other hand, the inactivation of Ku/LigD increased the efficiency of the deletion of G in the mutant strain.

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