scholarly journals Hyperactive mariner transposons are created by mutations that disrupt allosterism and increase the rate of transposon end synapsis

2013 ◽  
Vol 42 (4) ◽  
pp. 2637-2645 ◽  
Author(s):  
Danxu Liu ◽  
Ronald Chalmers
Keyword(s):  
Biochemical Analysis ◽  
Point Mutations ◽  
Double Strand Breaks ◽  
Amino Acid Motif ◽  
Transposase Gene ◽  
Strand Breaks ◽  
Dimer Interface ◽  
Almost All ◽  
New Applications ◽  
Increased Activity

Abstract New applications for transposons in vertebrate genetics have spurred efforts to develop hyperactive variants. Typically, a genetic screen is used to identify several hyperactive point mutations, which are then incorporated in a single transposase gene. However, the mechanisms responsible for the increased activity are unknown. Here we show that several point mutations in the mariner transposase increase their activities by disrupting the allostery that normally serves to downregulate transposition by slowing synapsis of the transposon ends. We focused on the conserved WVPHEL amino acid motif, which forms part of the mariner transposase dimer interface. We generated almost all possible single substitutions of the W, V, E and L residues and found that the majority are hyperactive. Biochemical analysis of the mutations revealed that they disrupt signals that pass between opposite sides of the developing transpososome in response to transposon end binding. In addition to their role in allostery, the signals control the initiation of catalysis, thereby preventing non-productive double-strand breaks. Finally, we note that such breaks may explain the puzzling ‘self-inflicted wounds’ at the ends of the Mos1 transposon in Drosophila.

scholarly journals TOPOVIBL-REC114 interaction regulates meiotic DNA double-strand breaks

2021 ◽  
Author(s):  
Alexandre Nore ◽  
Ariadna B Juarez-Martinez ◽  
Julie AJ Clement ◽  
Christine Brun ◽  
Bouboub Diagouraga ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Point Mutations ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Catalytic Complex ◽  
Strand Breaks ◽  
Genome Wide ◽  
Dna Cleavage Activity ◽  
Meiotic Dsbs

Meiosis requires the formation of programmed DNA double strand breaks (DSBs), essential for fertility and for generating genetic diversity. In male and female meiotic cells, DSBs are induced by the catalytic activity of the TOPOVIL complex formed by SPO11 and TOPOVIBL. To ensure genomic integrity, DNA cleavage activity is tightly regulated, and several accessory factors (REC114, MEI4, IHO1, and MEI1) are needed for DSB formation in mice. How and when these proteins act is not understood. Here, we show that REC114 is a direct partner of TOPOVIBL, and identified their conserved interacting domains by structural analysis. We then analysed the role of this interaction by monitoring meiotic DSBs in female and male mice carrying point mutations in TOPOVIBL that decrease or disrupt its binding to REC114. In these mutants, DSB activity was strongly reduced genome-wide in oocytes, but only in sub-telomeric regions in spermatocytes. In addition, in mutant spermatocytes, DSB activity was delayed in autosomes. These results provide evidence that REC114 is a key member of the TOPOVIL catalytic complex, and that the REC114/TOPOVIBL interaction ensures the efficiency and timing of DSB activity by integrating specific chromosomal features.

scholarly journals Indirect and direct evidence for DNA double–strand breaks in hypermutating immunoglobulin genes

2001 ◽  
Vol 356 (1405) ◽  
pp. 119-125 ◽  
Author(s):  
Heinz Jacobs ◽  
Klaus Rajewsky ◽  
Yosho Fukita ◽  
Linda Bross
Keyword(s):  
Somatic Hypermutation ◽  
Gene Segment ◽  
Point Mutations ◽  
Antigen Receptor ◽  
Double Strand Breaks ◽  
Mouse Strains ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Receptor Repertoire ◽  
Igh Locus

The generation of a diverse antigen receptor repertoire is fundamental for the functionality of the adaptive immune system. While the V(D)J recombination process that generates the primary antigen receptor repertoire is understood in great detail, it is still unclear by which mechanism immunoglobulin (Ig) genes are further diversified by somatic hypermutation. Using mouse strains that carry a non–functional, predefined V H D H J H gene segment in their IgH locus we demonstrate DNA double–strand breaks (DSBs) in and around V H D H J H in B cells undergoing somatic hypermutation. The generation of these DSBs depends on transcriptional activity, and their distribution along the V H D H J H segment parallels that of point mutations in the hypermutation domain. Furthermore, similar to hot spots of somatic hypermutation, 50–60% of all DSBs occur preferentially at RGYW motifs. DSBs may transiently dissociate the Ig promoter from the intronic enhancer to block further transcription and to initiate an error–prone nonhomologous DSB repair pathway. In accord with this model large deletions are frequently produced, along with point mutations, in a V H D H J H segment inserted together with its promoter into the IgH locus in inverted orientation. Our data suggest that DSBs are reaction intermediates of the mechanism underlying somatic hypermutation.

scholarly journals Homology Requirements for Targeting Heterologous Sequences During P-Induced Gap Repair in Drosophila melanogaster  1

Genetics ◽  
1997 ◽  
Vol 147 (2) ◽  
pp. 689-699 ◽  
Author(s):  
Tammy Dray ◽  
Gregory B Gloor
Keyword(s):  
Drosophila Melanogaster ◽  
Point Mutations ◽  
Double Strand Breaks ◽  
Yellow Gene ◽  
P Element ◽  
White Gene ◽  
Base Pairs ◽  
Strand Breaks ◽  
Distal Point ◽  
White Locus

The effect of homology on gene targeting was studied in the context of P-element-induced double-strand breaks at the white locus of Drosophila melanogaster. Double-strand breaks were made by excision of P-whd, a P-element insertion in the white gene. A nested set of repair templates was generated that contained the 8 kilobase (kb) yellow gene embedded within varying amounts of white gene sequence. Repair with unlimited homology was also analyzed. Flies were scored phenotypically for conversion of the yellow gene to the white locus. Targeting of the yellow gene was abolished when all of the 3′ homology was removed. Increases in template homology up to 51 base pairs (bp) did not significantly promote targeting. Maximum conversion was observed with a construct containing 493 bp of homology, without a significant increase in frequency when homology extended to the tips of the chromosome. These results demonstrate that the homology requirements for targeting a large heterologous insertion are quite different than those for a point mutation. Furthermore, heterologous insertions strongly affect the homology requirements for the conversion of distal point mutations. Several aberrant conversion tracts, which arose from templates that contained reduced homology, also were examined and characterized.

scholarly journals Genetic evidence for the involvement of mismatch repair proteins, PMS2 and MLH3, in a late step of homologous recombination

2020 ◽  
Vol 295 (51) ◽  
pp. 17460-17475
Author(s):  
Md Maminur Rahman ◽  
Mohiuddin Mohiuddin ◽  
Islam Shamima Keka ◽  
Kousei Yamada ◽  
Masataka Tsuda ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Mismatch Repair ◽  
Ectopic Expression ◽  
Point Mutations ◽  
Sister Chromatid Exchanges ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Γ Irradiation ◽  
Strand Breaks ◽  
Homologous Sequences

Homologous recombination (HR) repairs DNA double-strand breaks using intact homologous sequences as template DNA. Broken DNA and intact homologous sequences form joint molecules (JMs), including Holliday junctions (HJs), as HR intermediates. HJs are resolved to form crossover and noncrossover products. A mismatch repair factor, MLH3 endonuclease, produces the majority of crossovers during meiotic HR, but it remains elusive whether mismatch repair factors promote HR in nonmeiotic cells. We disrupted genes encoding the MLH3 and PMS2 endonucleases in the human B cell line, TK6, generating null MLH3−/− and PMS2−/− mutant cells. We also inserted point mutations into the endonuclease motif of MLH3 and PMS2 genes, generating endonuclease death MLH3DN/DN and PMS2EK/EK cells. MLH3−/− and MLH3DN/DN cells showed a very similar phenotype, a 2.5-fold decrease in the frequency of heteroallelic HR-dependent repair of restriction enzyme–induced double-strand breaks. PMS2−/− and PMS2EK/EK cells showed a phenotype very similar to that of the MLH3 mutants. These data indicate that MLH3 and PMS2 promote HR as an endonuclease. The MLH3DN/DN and PMS2EK/EK mutations had an additive effect on the heteroallelic HR. MLH3DN/DN/PMS2EK/EK cells showed normal kinetics of γ-irradiation–induced Rad51 foci but a significant delay in the resolution of Rad51 foci and a 3-fold decrease in the number of cisplatin-induced sister chromatid exchanges. The ectopic expression of the Gen1 HJ re-solvase partially reversed the defective heteroallelic HR of MLH3DN/DN/PMS2EK/EK cells. Taken together, we propose that MLH3 and PMS2 promote HR as endonucleases, most likely by processing JMs in mammalian somatic cells.

scholarly journals A Domesticated piggyBac Transposase Plays Key Roles in Heterochromatin Dynamics and DNA Cleavage during Programmed DNA Deletion in Tetrahymena thermophila

2010 ◽  
Vol 21 (10) ◽  
pp. 1753-1762 ◽  
Author(s):  
Chao-Yin Cheng ◽  
Alexander Vogt ◽  
Kazufumi Mochizuki ◽  
Meng-Chao Yao
Keyword(s):  
Dna Cleavage ◽  
Double Strand Breaks ◽  
Cellular Functions ◽  
Transposase Gene ◽  
Strand Breaks ◽  
Dna Deletion ◽  
Final Assembly ◽  
Dna Elimination ◽  
Cleavage Step

Transposons comprise large fractions of eukaryotic genomes and provide genetic reservoirs for the evolution of new cellular functions. We identified TPB2, a homolog of the piggyBac transposase gene that is required for programmed DNA deletion in Tetrahymena. TPB2 was expressed exclusively during the time of DNA excision, and its encoded protein Tpb2p was localized in DNA elimination heterochromatin structures. Notably, silencing of TPB2 by RNAi disrupts the final assembly of these heterochromatin structures and prevents DNA deletion to occur. In vitro studies revealed that Tpb2p is an endonuclease that produces double-strand breaks with four-base 5′ protruding ends, similar to the ends generated during DNA deletion. These findings suggest that Tpb2p plays a key role in the assembly of specialized DNA elimination chromatin architectures and is likely responsible for the DNA cleavage step of programmed DNA deletion.

scholarly journals Microhomology-mediated CRISPR/Cas9-based method for genome editing in fission yeast

2018 ◽  
Author(s):  
Aki Hayashi ◽  
Katsunori Tanaka
Keyword(s):  
Fission Yeast ◽  
Genome Editing ◽  
High Frequency ◽  
Target Genes ◽  
Point Mutations ◽  
Inducible Promoter ◽  
Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Guide Rna

The CRISPR/Cas9 system enables the editing of genomes of numerous organisms through the induction of the double-strand breaks (DSB) at specific chromosomal targets. We improved the CRISPR/Cas9 system to ease the direct introduction of a point mutation or a tagging sequence into the chromosome by combining it with the microhomology mediated end joining (MMEJ)-based genome editing in fission yeast. We constructed convenient cloning vectors, which possessed a guide RNA (gRNA) expression module, or the humanized Streptococcus pyogenes Cas9 gene that is expressed under the control of an inducible promoter to avoid the needless expression, or both a gRNA and Cas9 gene. Using this system, we attempted the MMEJ-mediated genome editing and found that the MMEJ-mediated method provides high-frequency genome editing at target loci without the need of a long donor DNA. Using short oligonucleotides, we successfully introduced point mutations into two target genes at high frequency. We also precisely integrated the sequences for epitope and GFP tagging using donor DNA possessing microhomology into the target loci, which enabled us to obtain cells expressing N-terminally tagged fusion proteins. This system could expedite genome editing in fission yeast, and could be applicable to other organisms.

scholarly journals Prime Editing for Inherited Retinal Diseases

2021 ◽  
Vol 3 ◽  
Author(s):  
Bruna Lopes da Costa ◽  
Sarah R. Levi ◽  
Eric Eulau ◽  
Yi-Ting Tsai ◽  
Peter M. J. Quinn
Keyword(s):  
Clinical Trials ◽  
De Novo ◽  
Point Mutations ◽  
Genetic Mutation ◽  
Double Strand Breaks ◽  
Retinal Diseases ◽  
Augmentation Therapy ◽  
Disease Etiology ◽  
Strand Breaks ◽  
Hereditary Disorders

Inherited retinal diseases (IRDs) are chronic, hereditary disorders that lead to progressive degeneration of the retina. Disease etiology originates from a genetic mutation—inherited or de novo—with a majority of IRDs resulting from point mutations. Given the plethora of IRDs, to date, mutations that cause these dystrophies have been found in approximately 280 genes. However, there is currently only one FDA-approved gene augmentation therapy, Luxturna (voretigene neparvovec-rzyl), available to patients with RPE65-mediated retinitis pigmentosa (RP). Although clinical trials for other genes are underway, these techniques typically involve gene augmentation rather than genome surgery. While gene augmentation therapy delivers a healthy copy of DNA to the cells of the retina, genome surgery uses clustered regularly interspaced short palindromic repeats (CRISPR)-based technology to correct a specific genetic mutation within the endogenous genome sequence. A new technique known as prime editing (PE) applies a CRISPR-based technology that possesses the potential to correct all twelve possible transition and transversion mutations as well as small insertions and deletions. EDIT-101, a CRISPR-based therapy that is currently in clinical trials, uses double-strand breaks and nonhomologous end joining to remove the IVS26 mutation in the CEP290 gene. Preferably, PE does not cause double-strand breaks nor does it require any donor DNA repair template, highlighting its unparalleled efficiency. Instead, PE uses reverse transcriptase and Cas9 nickase to repair mutations in the genome. While this technique is still developing, with several challenges yet to be addressed, it offers promising implications for the future of IRD treatment.

scholarly journals Heterologous synapsis in C. elegans is regulated by meiotic double-strand breaks and crossovers

2021 ◽  
Author(s):  
Hanwenheng Liu ◽  
Spencer G. Gordon ◽  
Ofer Rog
Keyword(s):  
Nuclear Envelope ◽  
Synaptonemal Complex ◽  
Meiotic Prophase ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
C Elegans ◽  
Homologous Sequences ◽  
Almost All ◽  
Break Formation ◽  
Successful Production

AbstractAlignment of the parental chromosomes during meiotic prophase is key to the formation of genetic exchanges, or crossovers, and consequently to the successful production of gametes. In almost all studied organisms, alignment involves synapsis: the assembly of a conserved inter-chromosomal interface called the synaptonemal complex (SC). While the SC usually synapses homologous sequences, it can assemble between heterologous sequences. However, little is known about the regulation of heterologous synapsis. Here we study the dynamics of heterologous synapsis in the nematode C. elegans. We characterize two experimental scenarios: SC assembly onto a folded-back chromosome that cannot pair with its homologous partner; and synapsis of pseudo-homologs, a fusion chromosome partnering with an unfused chromosome half its size. We observed elevated levels of heterologous synapsis when the number of meiotic double-strand breaks or crossovers were reduced, indicating that the promiscuity of synapsis is regulated by break formation or repair. By manipulating the levels of breaks and crossovers, we infer both chromosome-specific and nucleus-wide regulation on heterologous synapsis. Finally, we identify differences between the two conditions, suggesting that attachment to the nuclear envelope plays a role in regulating heterologous synapsis.

scholarly journals Structure of the catalytic domain of Mre11 fromChaetomium thermophilum

2015 ◽  
Vol 71 (6) ◽  
pp. 752-757 ◽  
Author(s):  
Florian Ulrich Seifert ◽  
Katja Lammens ◽  
Karl-Peter Hopfner
Keyword(s):  
Crystal Structure ◽  
Protein Complex ◽  
Catalytic Domain ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Dimer Interface ◽  
Chaetomium Thermophilum ◽  
Evolutionarily Conserved ◽  
Dna Ends

Together with the Rad50 ATPase, the Mre11 nuclease forms an evolutionarily conserved protein complex that plays a central role in the repair of DNA double-strand breaks (DSBs). Mre11–Rad50 detects and processes DNA ends, and has functions in the tethering as well as the signalling of DSBs. The Mre11 dimer can bind one or two DNA ends or hairpins, and processes DNA endonucleolytically as well as exonucleolytically in the 3′-to-5′ direction. Here, the crystal structure of the Mre11 catalytic domain dimer fromChaetomium thermophilum(CtMre11CD) is reported. CtMre11CDcrystals diffracted to 2.8 Å resolution and revealed previously undefined features within the dimer interface, in particular fully ordered eukaryote-specific insertion loops that considerably expand the dimer interface. Furthermore, comparison with other eukaryotic Mre11 structures reveals differences in the conformations of the dimer and the capping domain. In summary, the results reported here provide new insights into the architecture of the eukaryotic Mre11 dimer.

scholarly journals CRISPR base editing applications for identifying cancer-driving mutations

2021 ◽  
Author(s):  
Martin Pal ◽  
Marco J. Herold
Keyword(s):  
Point Mutations ◽  
Double Strand Breaks ◽  
Great Promise ◽  
Functional Screening ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Base Editing ◽  
Dna Base ◽  
Damage Response ◽  
Dna Template

CRISPR base editing technology is a promising genome editing tool as (i) it does not require a DNA template to introduce mutations and (ii) it avoids creating DNA double-strand breaks, which can lead to unintended chromosomal alterations or elicit an unwanted DNA damage response. Given many cancers originate from point mutations in cancer-driving genes, the application of base editing for either modelling tumour development, therapeutic editing, or functional screening is of great promise. In this review, we summarise current DNA base editing technologies and will discuss recent advancements and existing hurdles for its usage in cancer research.

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