Transient Silencing of DNA Repair Genes Improves Targeted Gene Integration in the Filamentous Fungus Trichoderma reesei

ABSTRACT Trichoderma reesei is a filamentous fungus that is used worldwide to produce industrial enzymes. Industrial strains have traditionally been created though systematic strain improvement using mutagenesis and screening approaches. It is also desirable to specifically manipulate the genes of the organism to further improve and to modify the strain. Targeted integration in filamentous fungi is typically hampered by very low frequencies of homologous recombination. To address this limitation, we have developed a simple transient method for silencing genes in T. reesei. Using gene-specific small interfering RNAs (siRNAs) targeted to mus53, we could achieve up to 90% knockdown of mus53 mRNA. As a practical example, we demonstrated that transient silencing of DNA repair genes significantly improved homologous integration of DNA at a specific locus in a standard protoplast transformation. The best transient silencing of mus53 with siRNAs in protoplasts could achieve up to 59% marker gene integration. IMPORTANCE The previous solution for improving targeted integration efficiency has been deleting nonhomologous end joining (NHEJ) DNA repair genes. However, deleting these important repair genes may lead to unintended consequences for genomic stability and could lead to the accumulation of spontaneous mutations. Our method of transiently silencing NHEJ repair pathway genes allows recovery of their important repair functions. Here we report a silencing approach for improving targeted DNA integration in filamentous fungi. Furthermore, our transient silencing method is a truly flexible approach that is capable of knocking down the expression of a target gene in growing mycelial cultures, which could facilitate the broad study of gene functions in T. reesei.

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Truncating variants in DNA-repair genes and their effect on AAO of hereditary breast cancer

10.1055/s-0038-1651801 ◽

2018 ◽

Author(s):

I Sepahi ◽

U Faust ◽

M Sturm ◽

K Bosse ◽

M Kehrer ◽

...

Keyword(s):

Breast Cancer ◽

Dna Repair ◽

Hereditary Breast Cancer ◽

Dna Repair Genes ◽

Repair Genes

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Comprehensive Analyses of Copy Number Variations In DNA Repair Genes Reveal the Role of Poly(ADP-Ribose) Polymerase in Longevity in Trees

SSRN Electronic Journal ◽

10.2139/ssrn.3751787 ◽

2020 ◽

Author(s):

Yuta Aoyagi Blue ◽

Junko Kusumi ◽

Akiko Satake

Keyword(s):

Dna Repair ◽

Copy Number ◽

Copy Number Variations ◽

Dna Repair Genes ◽

Repair Genes

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Copy number analyses of DNA repair genes reveal the role of poly(ADP-ribose) polymerase (PARP) in tree longevity

iScience ◽

10.1016/j.isci.2021.102779 ◽

2021 ◽

pp. 102779

Author(s):

Yuta Aoyagi Blue ◽

Junko Kusumi ◽

Akiko Satake

Keyword(s):

Dna Repair ◽

Copy Number ◽

Dna Repair Genes ◽

Repair Genes

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Coexistence of SOS-Dependent and SOS-Independent Regulation of DNA Repair Genes in Radiation-Resistant Deinococcus Bacteria

Cells ◽

10.3390/cells10040924 ◽

2021 ◽

Vol 10 (4) ◽

pp. 924

Author(s):

Laurence Blanchard ◽

Arjan de Groot

Keyword(s):

Dna Repair ◽

Deinococcus Radiodurans ◽

Dna Repair Genes ◽

Lesion Bypass ◽

Radiation Resistant ◽

Radiation Induced ◽

Induced Mutagenesis ◽

High Doses ◽

Translesion Polymerases ◽

Repair Genes

Deinococcus bacteria are extremely resistant to radiation and able to repair a shattered genome in an essentially error-free manner after exposure to high doses of radiation or prolonged desiccation. An efficient, SOS-independent response mechanism to induce various DNA repair genes such as recA is essential for radiation resistance. This pathway, called radiation/desiccation response, is controlled by metallopeptidase IrrE and repressor DdrO that are highly conserved in Deinococcus. Among various Deinococcus species, Deinococcus radiodurans has been studied most extensively. Its genome encodes classical DNA repair proteins for error-free repair but no error-prone translesion DNA polymerases, which may suggest that absence of mutagenic lesion bypass is crucial for error-free repair of massive DNA damage. However, many other radiation-resistant Deinococcus species do possess translesion polymerases, and radiation-induced mutagenesis has been demonstrated. At least dozens of Deinococcus species contain a mutagenesis cassette, and some even two cassettes, encoding error-prone translesion polymerase DnaE2 and two other proteins, ImuY and ImuB-C, that are probable accessory factors required for DnaE2 activity. Expression of this mutagenesis cassette is under control of the SOS regulators RecA and LexA. In this paper, we review both the RecA/LexA-controlled mutagenesis and the IrrE/DdrO-controlled radiation/desiccation response in Deinococcus.

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Patients with Systemic Sclerosis Present Increased DNA Damage Differentially Associated with DNA Repair Gene Polymorphisms

The Journal of Rheumatology ◽

10.3899/jrheum.130376 ◽

2014 ◽

Vol 41 (3) ◽

pp. 458-465 ◽

Cited By ~ 11

Author(s):

Gustavo Martelli Palomino ◽

Carmen L. Bassi ◽

Isabela J. Wastowski ◽

Danilo J. Xavier ◽

Yara M. Lucisano-Valim ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Systemic Sclerosis ◽

Blood Cells ◽

Gene Polymorphisms ◽

Peripheral Blood Cells ◽

Dna Repair Genes ◽

Repair Gene ◽

Dna Repair Gene ◽

Repair Genes

Objective.Patients with systemic sclerosis (SSc) exhibit increased toxicity when exposed to genotoxic agents. In our study, we evaluated DNA damage and polymorphic sites in 2 DNA repair genes (XRCC1Arg399Gln andXRCC4Ile401Thr) in patients with SSc.Methods.A total of 177 patients were studied for DNA repair gene polymorphisms. Fifty-six of them were also evaluated for DNA damage in peripheral blood cells using the comet assay.Results.Compared to controls, the patients as a whole or stratified into major clinical variants (limited or diffuse skin involvement), irrespective of the underlying treatment schedule, exhibited increased DNA damage.XRCC1(rs: 25487) andXRCC4(rs: 28360135) allele and genotype frequencies observed in patients with SSc were not significantly different from those observed in controls; however, theXRCC1Arg399Gln allele was associated with increased DNA damage only in healthy controls and theXRCC4Ile401Thr allele was associated with increased DNA damage in both patients and controls. Further, theXRCC1Arg399Gln allele was associated with the presence of antinuclear antibody and anticentromere antibody. No association was observed between these DNA repair gene polymorphic sites and clinical features of patients with SSc.Conclusion.These results corroborate the presence of genomic instability in SSc peripheral blood cells, as evaluated by increased DNA damage, and show that polymorphic sites of theXRCC1andXRCC4DNA repair genes may differentially influence DNA damage and the development of autoantibodies.

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