scholarly journals Efficient Gene Replacements in Toxoplasma gondii Strains Deficient for Nonhomologous End Joining

2009 ◽  
Vol 8 (4) ◽  
pp. 520-529 ◽  
Author(s):  
Barbara A. Fox ◽  
Jessica G. Ristuccia ◽  
Jason P. Gigley ◽  
David J. Bzik
Keyword(s):  
Dna Repair ◽  
Toxoplasma Gondii ◽  
Gene Targeting ◽  
Nonhomologous End Joining ◽  
Type I ◽  
Repair Pathway ◽  
Dna Breaks ◽  
End Joining ◽  
Γ Irradiation ◽  
Efficient Gene

ABSTRACT A high frequency of nonhomologous recombination has hampered gene targeting approaches in the model apicomplexan parasite Toxoplasma gondii. To address whether the nonhomologous end-joining (NHEJ) DNA repair pathway could be disrupted in this obligate intracellular parasite, putative KU proteins were identified and a predicted KU80 gene was deleted. The efficiency of gene targeting via double-crossover homologous recombination at several genetic loci was found to be greater than 97% of the total transformants in KU80 knockouts. Gene replacement efficiency was markedly increased (300- to 400-fold) in KU80 knockouts compared to wild-type strains. Target DNA flanks of only ∼500 bp were found to be sufficient for efficient gene replacements in KU80 knockouts. KU80 knockouts stably retained a normal growth rate in vitro and the high virulence phenotype of type I strains but exhibited an increased sensitivity to double-strand DNA breaks induced by treatment with phleomycin or γ-irradiation. Collectively, these results revealed that a significant KU-dependent NHEJ DNA repair pathway is present in Toxoplasma gondii. Integration essentially occurs only at the homologous targeted sites in the KU80 knockout background, making this genetic background an efficient host for gene targeting to speed postgenome functional analysis and genetic dissection of parasite biology.

scholarly journals MiR223-3p promotes synthetic lethality in BRCA1-deficient cancers

2019 ◽  
Vol 116 (35) ◽  
pp. 17438-17443 ◽  
Author(s):  
Gayathri Srinivasan ◽  
Elizabeth A. Williamson ◽  
Kimi Kong ◽  
Aruna S. Jaiswal ◽  
Guangcun Huang ◽  
...  
Keyword(s):  
Dna Repair ◽  
Cancer Cells ◽  
Synthetic Lethality ◽  
Negative Regulator ◽  
Nonhomologous End Joining ◽  
Chromosomal Translocations ◽  
Replication Forks ◽  
Repair Pathway ◽  
End Joining ◽  
Parp1 Inhibitors

Defects in DNA repair give rise to genomic instability, leading to neoplasia. Cancer cells defective in one DNA repair pathway can become reliant on remaining repair pathways for survival and proliferation. This attribute of cancer cells can be exploited therapeutically, by inhibiting the remaining repair pathway, a process termed synthetic lethality. This process underlies the mechanism of the Poly-ADP ribose polymerase-1 (PARP1) inhibitors in clinical use, which target BRCA1 deficient cancers, which is indispensable for homologous recombination (HR) DNA repair. HR is the major repair pathway for stressed replication forks, but when BRCA1 is deficient, stressed forks are repaired by back-up pathways such as alternative nonhomologous end-joining (aNHEJ). Unlike HR, aNHEJ is nonconservative, and can mediate chromosomal translocations. In this study we have found that miR223-3p decreases expression of PARP1, CtIP, and Pso4, each of which are aNHEJ components. In most cells, high levels of microRNA (miR) 223–3p repress aNHEJ, decreasing the risk of chromosomal translocations. Deletion of the miR223 locus in mice increases PARP1 levels in hematopoietic cells and enhances their risk of unprovoked chromosomal translocations. We also discovered that cancer cells deficient in BRCA1 or its obligate partner BRCA1-Associated Protein-1 (BAP1) routinely repress miR223-3p to permit repair of stressed replication forks via aNHEJ. Reconstituting the expression of miR223-3p in BRCA1- and BAP1-deficient cancer cells results in reduced repair of stressed replication forks and synthetic lethality. Thus, miR223-3p is a negative regulator of the aNHEJ DNA repair and represents a therapeutic pathway for BRCA1- or BAP1-deficient cancers.

scholarly journals The C-Terminal Domain of Cernunnos/XLF Is Dispensable for DNA Repair In Vivo

2008 ◽  
Vol 29 (5) ◽  
pp. 1116-1122 ◽  
Author(s):  
Laurent Malivert ◽  
Isabelle Callebaut ◽  
Paola Rivera-Munoz ◽  
Alain Fischer ◽  
Jean-Paul Mornon ◽  
...  
Keyword(s):  
Dna Repair ◽  
Nonhomologous End Joining ◽  
Dna Ligase ◽  
Repair Pathway ◽  
End Joining ◽  
Functional Assays ◽  
The Core ◽  
Ligase Iv

ABSTRACT The core nonhomologous end-joining DNA repair pathway is composed of seven factors: Ku70, Ku80, DNA-PKcs, Artemis, XRCC4 (X4), DNA ligase IV (L4), and Cernunnos/XLF (Cernunnos). Although Cernunnos and X4 are structurally related and participate in the same complex together with L4, they have distinct functions during DNA repair. L4 relies on X4 but not on Cernunnos for its stability, and L4 is required for optimal interaction of Cernunnos with X4. We demonstrate here, using in vitro-generated Cernunnos mutants and a series of functional assays in vivo, that the C-terminal region of Cernunnos is dispensable for its activity during DNA repair.

scholarly journals Evidence that Stable Retroviral Transduction and Cell Survival following DNA Integration Depend on Components of the Nonhomologous End Joining Repair Pathway

2004 ◽  
Vol 78 (16) ◽  
pp. 8573-8581 ◽  
Author(s):  
René Daniel ◽  
James G. Greger ◽  
Richard A. Katz ◽  
Konstantin D. Taganov ◽  
Xiaoyun Wu ◽  
...  
Keyword(s):  
Dna Repair ◽  
Nonhomologous End Joining ◽  
Transduction Efficiency ◽  
Repair Pathway ◽  
Integration Process ◽  
End Joining ◽  
General Role ◽  
Dna Integration ◽  
Ligase Iv ◽  
Hiv 1

ABSTRACT We have previously reported several lines of evidence that support a role for cellular DNA repair systems in completion of the retroviral DNA integration process. Failure to repair an intermediate in the process of integrating viral DNA into host DNA appears to trigger growth arrest or death of a large percentage of infected cells. Cellular proteins involved in the nonhomologous end joining (NHEJ) pathway (DNA-PKCS) and the damage-signaling kinases (ATM and ATR) have been implicated in this process. However, some studies have suggested that NHEJ proteins may not be required for the completion of lentiviral DNA integration. Here we provide additional evidence that NHEJ proteins are required for stable transduction by human immunodeficiency type 1 (HIV-1)-based vectors. Our analyses with two different reporters show that the number of stably transduced DNA-PKCS-deficient scid fibroblasts was reduced by 80 to 90% compared to the number of control cells. Furthermore, transduction efficiency can be restored to wild-type levels in scid cells that are complemented with a functional DNA-PKCS gene. The efficiency of stable transduction by an HIV-1-based vector is also reduced upon infection of Xrcc4 and ligase IV-deficient cells, implying a role for these components of the NHEJ repair pathway. Finally, we show that cells deficient in ligase IV are killed by infection with an integrase-competent but not an integrase-deficient HIV-1 vector. Results presented in this study lend further support to a general role for the NHEJ DNA repair pathway in completion of the retroviral DNA integration process.

scholarly journals Nonhomologous-End-Joining Factors Regulate DNA Repair Fidelity during Sleeping Beauty Element Transposition in Mammalian Cells

2003 ◽  
Vol 23 (23) ◽  
pp. 8505-8518 ◽  
Author(s):  
Stephen R. Yant ◽  
Mark A. Kay
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Mammalian Cells ◽  
Donor Site ◽  
Protective Role ◽  
Sleeping Beauty ◽  
Nonhomologous End Joining ◽  
Dna Breaks ◽  
End Joining ◽  
Transposon Insertion

ABSTRACT Herein, we report that the DNA-dependent protein kinase (DNA-PK) regulates the DNA damage introduced during Sleeping Beauty (SB) element excision and reinsertion in mammalian cells. Using both plasmid- and chromosome-based mobility assays, we analyzed the repair of transposase-induced double-stranded DNA breaks in cells deficient in either the DNA-binding subunit of DNA-PK (Ku) or its catalytic subunit (DNA-PKcs). We found that the free 3′ overhangs left after SB element excision were efficiently and accurately processed by the major Ku-dependent nonhomologous-end-joining pathway. Rejoining of broken DNA molecules in the absence of Ku resulted in extensive end degradation at the donor site and greatly increased the frequency of recombination with ectopic templates. Therefore, the major DNA-PK-dependent DNA damage response predominates over more-error-prone repair pathways and thereby facilitates high-fidelity DNA repair during transposon mobilization in mammalian cells. Although transposable elements were not found to be efficiently circularized after transposase-mediated excision, DNA-PK deficiency supported more-frequent transposase-mediated element insertion than was found in wild-type controls. We conclude that, based on its ability to regulate excision site junctional diversity and transposon insertion frequency, DNA-PK serves an important protective role during transpositional recombination in mammals.

scholarly journals Fixing DNA breaks during class switch recombination

2008 ◽  
Vol 205 (3) ◽  
pp. 509-513 ◽  
Author(s):  
Christopher J. Jolly ◽  
Adam J.L. Cook ◽  
John P. Manis
Keyword(s):  
Dna Sequences ◽  
Class Switch Recombination ◽  
Nonhomologous End Joining ◽  
Repair Pathway ◽  
Dna Breaks ◽  
End Joining ◽  
Dependent Protein Kinase ◽  
Class Switch ◽  
Dependent Protein

Immunoglobulin (Ig) class switch recombination (CSR) involves the breakage and subsequent repair of two DNA sequences, known as switch (S) regions, which flank IgH constant region exons. The resolution of CSR-associated breaks is thought to require the nonhomologous end-joining (NHEJ) DNA repair pathway, but the role of the NHEJ factor DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in this process has been unclear. A new study, in which broken IgH-containing chromosomes in switching B cells were visualized directly, clearly demonstrated that DNA-PKcs and, unexpectedly, the nuclease Artemis are involved in the resolution of switch breaks.

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