Harnessing mutagenic homologous recombination for targeted mutagenesis in vivo by TaGTEAM

AbstractTo clarify whether differential compartmentalization of Survivin impacts temozolomide (TMZ)-triggered end points, we established a well-defined glioblastoma cell model in vitro (LN229 and A172) and in vivo, distinguishing between its nuclear and cytoplasmic localization. Expression of nuclear export sequence (NES)-mutated Survivin (SurvNESmut-GFP) led to impaired colony formation upon TMZ. This was not due to enhanced cell death but rather due to increased senescence. Nuclear-trapped Survivin reduced homologous recombination (HR)-mediated double-strand break (DSB) repair, as evaluated by γH2AX foci formation and qPCR-based HR assay leading to pronounced induction of chromosome aberrations. Opposite, clones, expressing free-shuttling cytoplasmic but not nuclear-trapped Survivin, could repair TMZ-induced DSBs and evaded senescence. Mass spectrometry-based interactomics revealed, however, no direct interaction of Survivin with any of the repair factors. The improved TMZ-triggered HR activity in Surv-GFP was associated with enhanced mRNA and stabilized RAD51 protein expression, opposite to diminished RAD51 expression in SurvNESmut cells. Notably, cytoplasmic Survivin could significantly compensate for the viability under RAD51 knockdown. Differential Survivin localization also resulted in distinctive TMZ-triggered transcriptional pathways, associated with senescence and chromosome instability as shown by global transcriptome analysis. Orthotopic LN229 xenografts, expressing SurvNESmut exhibited diminished growth and increased DNA damage upon TMZ, as manifested by PCNA and γH2AX foci expression, respectively, in brain tissue sections. Consequently, those mice lived longer. Although tumors of high-grade glioma patients expressed majorly nuclear Survivin, they exhibited rarely NES mutations which did not correlate with survival. Based on our in vitro and xenograft data, Survivin nuclear trapping would facilitate glioma response to TMZ.

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Use of an Endogenous Plasmid Locus for Stable intransComplementation in Borrelia burgdorferi

Applied and Environmental Microbiology ◽

10.1128/aem.03657-14 ◽

2014 ◽

Vol 81 (3) ◽

pp. 1038-1046 ◽

Cited By ~ 5

Author(s):

Irene N. Kasumba ◽

Aaron Bestor ◽

Kit Tilly ◽

Patricia A. Rosa

Keyword(s):

Borrelia Burgdorferi ◽

Shuttle Vector ◽

Targeted Mutagenesis ◽

Multiple Cloning Site ◽

Stable Gene ◽

Content Type ◽

Shuttle Vectors ◽

Stable Maintenance

ABSTRACTTargeted mutagenesis and complementation are important tools for studying genes of unknown function in the Lyme disease spirocheteBorrelia burgdorferi. A standard method of complementation is reintroduction of a wild-type copy of the targeted gene on a shuttle vector. However, shuttle vectors are present at higher copy numbers thanB. burgdorferiplasmids and are potentially unstable in the absence of selection, thereby complicating analyses in the mouse-tick infectious cycle.B. burgdorferihas over 20 plasmids, with some, such as linear plasmid 25 (lp25), carrying genes required by the spirochetein vivobut relatively unstable duringin vitrocultivation. We propose that complementation on an endogenous plasmid such as lp25 would overcome the copy number andin vivostability issues of shuttle vectors. In addition, insertion of a selectable marker on lp25 could ensure its stable maintenance by spirochetes in culture. Here, we describe the construction of a multipurpose allelic-exchange vector containing a multiple-cloning site and either of two selectable markers. This suicide vector directs insertion of the complementing gene into thebbe02locus, a site on lp25 that was previously shown to be nonessential during bothin vitroandin vivogrowth. We demonstrate the functional utility of this strategy by restoring infectivity to anospCmutant through complementation at this site on lp25 and stable maintenance of theospCgene throughout mouse infection. We conclude that this represents a convenient and widely applicable method for stable gene complementation inB. burgdorferi.

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Rad51 catalytic mutants differentially affect the Rad51 nucleoprotein filament in vivo

10.1101/070276 ◽

2016 ◽

Author(s):

Maureen M. Mundia ◽

Alissa C. Magwood ◽

Mark D. Baker

Keyword(s):

Homologous Recombination ◽

Dna Synthesis ◽

Cell Lines ◽

Atp Hydrolysis ◽

Dominant Negative ◽

Strand Exchange ◽

Wild Type ◽

Hybridoma Cell Lines ◽

Insight Into

ABSTRACTIn this study, we utilized mouse hybridoma cell lines stably expressing ectopic wild-type Rad51, or the Rad51-K133A and Rad51-K133R catalytic mutants deficient in ATP binding and ATP hydrolysis, respectively, to investigate effects on the Rad51 nucleoprotein filament in vivo. Immunoprecipitation studies reveal interactions between ectopic wild-type Rad51, Rad51-K133A and Rad51-K133R and endogenous Rad51, Brca2 and p53 proteins. Importantly, the expression of Rad51-K133A and Rad51-K133R catalytic mutants (but not wild-type Rad51) targets endogenous Rad51, Brca2 and p53 proteins for proteasome-mediated degradation. Expression of Rad51-K133R significantly reduces nascent DNA synthesis (3’ polymerization) during homologous recombination (HR), but the effects of Rad51-K133A on 3’ polymerization are considerably more severe. Provision of additional wild-type Rad51 in cell lines expressing Rad51-K133A or Rad51-K133R does not restore diminished levels of endogenous Brca2, Rad51 or p53, nor restore the deficiency in 3’ polymerization. Cells expressing Rad51-K133A are also significantly reduced in their capacity to drive strand exchange through regions of heterology. Our results reveal an interesting mechanistic dichotomy in the way mutant Rad51-K133A and Rad51-K133R proteins influence 3’ polymerization and provide novel insight into the mechanism of their dominant-negative phenotypes.

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Chi-Dependent Intramolecular Recombination in Escherichia coli

Genetics ◽

10.1093/genetics/148.2.545 ◽

1998 ◽

Vol 148 (2) ◽

pp. 545-557

Author(s):

Rachel Friedman-Ohana ◽

Iris Karunker ◽

Amikam Cohen

Keyword(s):

Escherichia Coli ◽

Homologous Recombination ◽

Experimental System ◽

Cis Acting ◽

Enzymes Activity ◽

Intramolecular Recombination ◽

Insight Into

Abstract Homologous recombination in Escherichia coli is enhanced by a cis-acting octamer sequence named Chi (5′-GCTGGTGG-3′) that interacts with RecBCD. To gain insight into the mechanism of Chi-enhanced recombination, we recruited an experimental system that permits physical monitoring of intramolecular recombination by linear substrates released by in vivo restriction from infecting chimera phage. Recombination of the released substrates depended on recA, recBCD and cis-acting Chi octamers. Recombination proficiency was lowered by a xonA mutation and by mutations that inactivated the RuvABC and RecG resolution enzymes. Activity of Chi sites was influenced by their locations and by the number of Chi octamers at each site. A single Chi site stimulated recombination, but a combination of Chi sites on the two homologs was synergistic. These data suggest a role for Chi at both ends of the linear substrate. Chi was lost in all recombinational exchanges stimulated by a single Chi site. Exchanges in substrates with Chi sites on both homologs occurred in the interval between the sites as well as in the flanking interval. These observations suggest that the generation of circular products by intramolecular recombination involves Chi-dependent processing of one end by RecBCD and pairing of the processed end with its duplex homolog.

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An Efficient Site-Directed Mutagenesis Method In Vivo in Escherichia Coli

Applied Mechanics and Materials ◽

10.4028/www.scientific.net/amm.195-196.407 ◽

2012 ◽

Vol 195-196 ◽

pp. 407-411

Author(s):

Mu Qing Qiu

Keyword(s):

Escherichia Coli ◽

Homologous Recombination ◽

Recombinant Plasmid ◽

Genomic Dna ◽

Gene Replacement ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis

In order to develop an efficient site-directed mutagenesis method in vivo, the tests were tested by the following methods. The methods that the fragment knockouted ompR gene was constructed through overlapping PCR, digested by Notand Sal, ligated to plasmid pKOV were applied. The recombination plasmid was transformed into Escherichia coli WMC-001 strain, integrated into the genomic DNA through two step homologous recombination. The Escherichia coli WMC-001/ompR-mutant was obtained due to gene replacement. The fragment of the mutant ompR gene was amplified through overlapping PCR, cloned into pKOV vector. The recombinant plasmid was introduced into Escherichia coli WMC-001/ompR-mutant. The Escherichia coli WMC-001/ompR mutant was also obtained due to gene replacement. Results: The site-directed mutagenesis has been successfully constructed in the ompR gene by sequencing. Conclusion: The method is effective for construction of gene site-directed mutagenesis in vivo.

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SIRT1 contributes to telomere maintenance and augments global homologous recombination

The Journal of Cell Biology ◽

10.1083/jcb.201005160 ◽

2010 ◽

Vol 191 (7) ◽

pp. 1299-1313 ◽

Cited By ~ 156

Author(s):

Jose A. Palacios ◽

Daniel Herranz ◽

Maria Luigia De Bonis ◽

Susana Velasco ◽

Manuel Serrano ◽

...

Keyword(s):

Homologous Recombination ◽

Telomerase Activity ◽

Histone Deacetylation ◽

Telomere Maintenance ◽

Loss Of Function ◽

Entire Genome ◽

Telomere Shortening ◽

Telomere Biology ◽

Sir Complex

Yeast Sir2 deacetylase is a component of the silent information regulator (SIR) complex encompassing Sir2/Sir3/Sir4. Sir2 is recruited to telomeres through Rap1, and this complex spreads into subtelomeric DNA via histone deacetylation. However, potential functions at telomeres for SIRT1, the mammalian orthologue of yeast Sir2, are less clear. We studied both loss of function (SIRT1 deficient) and gain of function (SIRT1super) mouse models. Our results indicate that SIRT1 is a positive regulator of telomere length in vivo and attenuates telomere shortening associated with aging, an effect dependent on telomerase activity. Using chromatin immunoprecipitation assays, we find that SIRT1 interacts with telomeric repeats in vivo. In addition, SIRT1 overexpression increases homologous recombination throughout the entire genome, including telomeres, centromeres, and chromosome arms. These findings link SIRT1 to telomere biology and global DNA repair and provide new mechanistic explanations for the known functions of SIRT1 in protection from DNA damage and some age-associated pathologies.

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Using the Endogenous CRISPR-Cas System of Heliobacterium modesticaldum To Delete the Photochemical Reaction Center Core Subunit Gene

Applied and Environmental Microbiology ◽

10.1128/aem.01644-19 ◽

2019 ◽

Vol 85 (23) ◽

Cited By ~ 3

Author(s):

Patricia L. Baker ◽

Gregory S. Orf ◽

Kimberly Kevershan ◽

Michael E. Pyne ◽

Taner Bicer ◽

...

Keyword(s):

Homologous Recombination ◽

Genome Editing ◽

Reaction Center ◽

Photochemical Reaction ◽

Genetic Locus ◽

Gene Replacement ◽

Type I ◽

Content Type ◽

Heliobacterium Modesticaldum

ABSTRACT In Heliobacterium modesticaldum, as in many Firmicutes, deleting genes by homologous recombination using standard techniques has been extremely difficult. The cells tend to integrate the introduced plasmid into the chromosome by a single recombination event rather than perform the double recombination required to replace the targeted locus. Transformation with a vector containing only a homologous recombination template for replacement of the photochemical reaction center gene pshA produced colonies with multiple genotypes, rather than a clean gene replacement. To address this issue, we required an additional means of selection to force a clean gene replacement. In this study, we report the genetic structure of the type I-A and I-E CRISPR-Cas systems from H. modesticaldum, as well as methods to leverage the type I-A system for genome editing. In silico analysis of the CRISPR spacers revealed a potential consensus protospacer adjacent motif (PAM) required for Cas3 recognition, which was then tested using an in vivo interference assay. Introduction of a homologous recombination plasmid that carried a miniature CRISPR array targeting sequences in pshA (downstream of a naturally occurring PAM sequence) produced nonphototrophic transformants with clean replacements of the pshA gene with ∼80% efficiency. Mutants were confirmed by PCR, sequencing, optical spectroscopy, and growth characteristics. This methodology should be applicable to any genetic locus in the H. modesticaldum genome. IMPORTANCE The heliobacteria are the only phototrophic members of the largely Gram-positive phylum Firmicutes, which contains medically and industrially important members, such as Clostridium difficile and Clostridium acetobutylicum. Heliobacteria are of interest in the study of photosynthesis because their photosynthetic system is unique and the simplest known. Since their discovery in the early 1980s, work on the heliobacteria has been hindered by the lack of a genetic transformation system. The problem of introducing foreign DNA into these bacteria has been recently rectified by our group; however, issues still remained for efficient genome editing. The significance of this work is that we have characterized the endogenous type I CRISPR-Cas system in the heliobacteria and leveraged it to assist in genome editing. Using the CRISPR-Cas system allowed us to isolate transformants with precise replacement of the pshA gene encoding the main subunit of the photochemical reaction center.

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Targeted Mutagenesis of Dengue Virus Type 2 Replicon RNA by Yeast In Vivo Recombination

Dengue - Methods in Molecular Biology ◽

10.1007/978-1-4939-0348-1_10 ◽

2014 ◽

pp. 151-160

Author(s):

Mark Manzano ◽

Radhakrishnan Padmanabhan

Keyword(s):

Dengue Virus ◽

Virus Type ◽

Targeted Mutagenesis ◽

In Vivo Recombination ◽

Dengue Virus Type 2

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The internal region of CtIP negatively regulates DNA end resection

Nucleic Acids Research ◽

10.1093/nar/gkaa273 ◽

2020 ◽

Vol 48 (10) ◽

pp. 5485-5498 ◽

Cited By ~ 2

Author(s):

Sean Michael Howard ◽

Ilaria Ceppi ◽

Roopesh Anand ◽

Roger Geiger ◽

Petr Cejka

Keyword(s):

Homologous Recombination ◽

Regulatory Function ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Internal Region ◽

Dna End Resection ◽

End Resection

Abstract DNA double-strand breaks are repaired by end-joining or homologous recombination. A key-committing step of recombination is DNA end resection. In resection, phosphorylated CtIP first promotes the endonuclease of MRE11–RAD50–NBS1 (MRN). Subsequently, CtIP also stimulates the WRN/BLM–DNA2 pathway, coordinating thus both short and long-range resection. The structure of CtIP differs from its orthologues in yeast, as it contains a large internal unstructured region. Here, we conducted a domain analysis of CtIP to define the function of the internal region in DNA end resection. We found that residues 350–600 were entirely dispensable for resection in vitro. A mutant lacking these residues was unexpectedly more efficient than full-length CtIP in DNA end resection and homologous recombination in vivo, and consequently conferred resistance to lesions induced by the topoisomerase poison camptothecin, which require high MRN–CtIP-dependent resection activity for repair. This suggested that the internal CtIP region, further mapped to residues 550–600, may mediate a negative regulatory function to prevent over resection in vivo. The CtIP internal deletion mutant exhibited sensitivity to other DNA-damaging drugs, showing that upregulated resection may be instead toxic under different conditions. These experiments together identify a region within the central CtIP domain that negatively regulates DNA end resection.

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