Variation among Metschnikowia pulcherrima Isolates for Genetic Modification and Homologous Recombination

Metschnikowia pulcherrima is a non-conventional yeast with the potential to be used in biotechnological processes, especially involving low-cost feedstock exploitation. However, there are a lack of tools for researching it at a molecular level and for producing genetically modified strains. We tested the amenability to genetic modification of ten different strains, establishing a transformation protocol based on LiAc/PEG that allows us to introduce heterologous DNA. Non-homologous integration was broadly successful and homologous recombination was successful in two strains. Chemical inhibition of non-homologous end joining recombination had a modest effect on the improvement of homologous recombination rates. Removal of selective markers via flippase recombinase was successful across integrated loci except for those targeted to the native URA3 locus, suggesting that the genome sequence or structure alters the efficacy of this system.

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Analysis of chromatid-break-repair detects a homologous recombination to non-homologous end-joining switch with increasing load of DNA double-strand breaks

Mutation Research/Genetic Toxicology and Environmental Mutagenesis ◽

10.1016/j.mrgentox.2021.503372 ◽

2021 ◽

pp. 503372

Author(s):

Tamara Murmann-Konda ◽

Aashish Soni ◽

Martin Stuschke ◽

George Iliakis

Keyword(s):

Homologous Recombination ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Chromatid Break ◽

Break Repair ◽

Non Homologous End Joining ◽

Increasing Load

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Proton Irradiation Increases the Necessity for Homologous Recombination Repair Along with the Indispensability of Non-Homologous End Joining

Cells ◽

10.3390/cells9040889 ◽

2020 ◽

Vol 9 (4) ◽

pp. 889 ◽

Cited By ~ 3

Author(s):

Klaudia Szymonowicz ◽

Adam Krysztofiak ◽

Jansje van der Linden ◽

Ajvar Kern ◽

Simon Deycmar ◽

...

Keyword(s):

Dna Damage ◽

Homologous Recombination ◽

Proton Irradiation ◽

Negative Impact ◽

Particle Therapy ◽

Homologous Recombination Repair ◽

End Joining ◽

X Ray ◽

Recombination Repair ◽

Non Homologous End Joining

Technical improvements in clinical radiotherapy for maximizing cytotoxicity to the tumor while limiting negative impact on co-irradiated healthy tissues include the increasing use of particle therapy (e.g., proton therapy) worldwide. Yet potential differences in the biology of DNA damage induction and repair between irradiation with X-ray photons and protons remain elusive. We compared the differences in DNA double strand break (DSB) repair and survival of cells compromised in non-homologous end joining (NHEJ), homologous recombination repair (HRR) or both, after irradiation with an equal dose of X-ray photons, entrance plateau (EP) protons, and mid spread-out Bragg peak (SOBP) protons. We used super-resolution microscopy to investigate potential differences in spatial distribution of DNA damage foci upon irradiation. While DNA damage foci were equally distributed throughout the nucleus after X-ray photon irradiation, we observed more clustered DNA damage foci upon proton irradiation. Furthermore, deficiency in essential NHEJ proteins delayed DNA repair kinetics and sensitized cells to both, X-ray photon and proton irradiation, whereas deficiency in HRR proteins sensitized cells only to proton irradiation. We assume that NHEJ is indispensable for processing DNA DSB independent of the irradiation source, whereas the importance of HRR rises with increasing energy of applied irradiation.

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A role for the p53 tumour suppressor in regulating the balance between homologous recombination and non-homologous end joining

Open Biology ◽

10.1098/rsob.160225 ◽

2016 ◽

Vol 6 (9) ◽

pp. 160225 ◽

Cited By ~ 16

Author(s):

Sylvie Moureau ◽

Janna Luessing ◽

Emma Christina Harte ◽

Muriel Voisin ◽

Noel Francis Lowndes

Keyword(s):

Dna Damage ◽

Homologous Recombination ◽

Tumour Suppressor ◽

Cycle Phase ◽

Repair Pathway ◽

End Joining ◽

Dsb Repair ◽

Pathway Choice ◽

Non Homologous End Joining ◽

End Resection

Loss of p53, a transcription factor activated by cellular stress, is a frequent event in cancer. The role of p53 in tumour suppression is largely attributed to cell fate decisions. Here, we provide evidence supporting a novel role for p53 in the regulation of DNA double-strand break (DSB) repair pathway choice. 53BP1, another tumour suppressor, was initially identified as p53 Binding Protein 1, and has been shown to inhibit DNA end resection, thereby stimulating non-homologous end joining (NHEJ). Yet another tumour suppressor, BRCA1, reciprocally promotes end resection and homologous recombination (HR). Here, we show that in both human and mouse cells, the absence of p53 results in impaired 53BP1 focal recruitment to sites of DNA damage induced by ionizing radiation. This effect is largely independent of cell cycle phase and the extent of DNA damage. In p53-deficient cells, diminished localization of 53BP1 is accompanied by a reciprocal increase in BRCA1 recruitment to DSBs. Consistent with these findings, we demonstrate that DSB repair via NHEJ is abrogated, while repair via homology-directed repair (HDR) is stimulated. Overall, we propose that in addition to its role as an ‘effector’ protein in the DNA damage response, p53 plays a role in the regulation of DSB repair pathway choice.

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