Factors Affecting Double-Strand Break-Induced Homologous Recombination in Mammalian Cells

Pérez, C.; Guyot, Valérie; Cabaniols, Jean-Pierre; Gouble, Agnès; Micheaux, Beatrice; Smith, Justin K.; Leduc, Sophie; Pâques, Frédéric; Duchâteau, Philippe

doi:10.2144/05391gt01

Cited by 32 publications

(24 citation statements)

References 31 publications

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“…Transgenic homing endonucleases have been used extensively to study the repair of DSBs in nuclear chromosomes (16,31,32), but this reported usage for a cell organelle is unique. Transgenic restriction endonucleases have been used to damage mitochondrial DNA in animal cells (33), presumably because a homing enzyme target could not be introduced.…”

Section: Discussionmentioning

confidence: 99%

Microhomology-mediated and nonhomologous repair of a double-strand break in the chloroplast genome of Arabidopsis

Kwon

Huq

Herrin

2010

Proc. Natl. Acad. Sci. U.S.A.

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Chloroplast DNA (cpDNA) is under great photooxidative stress, yet its evolution is very conservative compared with nuclear or mitochondrial genomes. It can be expected that DNA repair mechanisms play important roles in cpDNA survival and evolution, but they are poorly understood. To gain insight into how the most severe form of DNA damage, a double-strand break (DSB), is repaired, we have developed an inducible system in Arabidopsis that employs a psbA intron endonuclease from Chlamydomonas, I-CreII, that is targeted to the chloroplast using the rbcS1 transit peptide. In Chlamydomonas, an I-CreII-induced DSB in psbA was repaired, in the absence of the intron, by homologous recombination between repeated sequences (20-60 bp) abundant in that genome; Arabidopsis cpDNA is very repeat poor, however. Phenotypically strong and weak transgenic lines were examined and shown to correlate with I-CreII expression levels. Southern blot hybridizations indicated a substantial loss of DNA at the psbA locus, but not cpDNA as a whole, in the strongly expressing line. PCR analysis identified deletions nested around the I-CreII cleavage site indicative of DSB repair using microhomology (6-12 bp perfect repeats, or 10-16 bp with mismatches) and no homology. These results provide evidence of alternative DSB repair pathways in the Arabidopsis chloroplast that resemble the nuclear, microhomologymediated and nonhomologous end joining pathways, in terms of the homology requirement. Moreover, when taken together with the results from Chlamydomonas, the data suggest an evolutionary relationship may exist between the repeat structure of the genome and the organelle's ability to repair broken chromosomes.DNA repair | evolution | homing endonuclease | I-CreII | plastid DNA C hloroplast DNA (cpDNA) is closely associated with the photosynthetic membranes that harvest radiant energy and strip electrons from water, producing molecular oxygen as a byproduct (1, 2). Highly reactive forms of oxygen are also generated, and despite the presence of detoxifying enzymes, photooxidative damage is a serious problem. Although there has been extensive study of the damage, protection, and repair of photosynthetic membranes, there has been little attention paid to the genetic consequences of photooxidative stress (3). This can be attributed, at least in part, to a grossly incomplete knowledge of how cpDNA is replicated and maintained throughout the life of a plant (3, 4).However, despite the high-stress environment, the evolution of cpDNA is more conservative than nuclear or mitochondrial DNA (5). We can thus infer that DNA repair mechanisms play important roles in protecting cpDNA and are intimately involved in its evolution. And although some processes have been identified (6-11), the panoply of organelle repair mechanisms is not known, nor is it clear which are most important, or how they have coevolved with the genome. Although we expect that some cpDNA repair processes will reflect its prokaryotic ancestry, the consequences of eukaryotic cell integration...

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Section: Discussionmentioning

confidence: 99%

Microhomology-mediated and nonhomologous repair of a double-strand break in the chloroplast genome of Arabidopsis

Kwon

Huq

Herrin

2010

Proc. Natl. Acad. Sci. U.S.A.

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“…Different species have different numbers of Rad51 paralogues and the importance of the Rad51 paralogues varies among different species. In higher eukaryotes, a Rad59-like protein has not yet been identified, despite having an SSA pathway that is surprisingly similar to the one in S. cerevisiae (44). However, the RAD51-independent pathway has not yet been thoroughly studied, and the existence of an as yet unidentified Rad52 paralogue important to SSA in higher eukaryotes remains a possibility.…”

Section: Rad59 Functions As a Rad52mentioning

confidence: 99%

DNA Annealing Mediated by Rad52 and Rad59 Proteins

Sugiyama²,

Kowalczykowski

2006

Journal of Biological Chemistry

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In the budding yeast Saccharomyces cerevisiae, the RAD52 gene is essential for all homologous recombination events and its homologue, the RAD59 gene, is important for those that occur independently of RAD51. Both Rad52 and Rad59 proteins can anneal complementary single-stranded (ss) DNA. We quantitatively examined the ssDNA annealing activity of Rad52 and Rad59 proteins and found significant differences in their biochemical properties. First, and most importantly, they differ in their ability to anneal ssDNA that is complexed with replication protein A (RPA). Rad52 can anneal an RPA-ssDNA complex, but Rad59 cannot. Second, Rad59-promoted DNA annealing follows first-order reaction kinetics, whereas Rad52-promoted annealing follows second-order reaction kinetics. Last, Rad59 enhances Rad52-mediated DNA annealing at increased NaCl concentrations, both in the absence and presence of RPA. These results suggest that Rad59 performs different functions in the recombination process, and should be more accurately viewed as a Rad52 paralogue.Repair of DNA double-strand breaks (DSBs) 3 is a crucial process needed to maintain cell viability and genomic integrity. An unrepaired DSB can result in growth arrest, loss of genetic information, or cell death (1). In Saccharomyces cerevisiae, genes in the RAD52 epistasis group are responsible for homologous recombination-dependent DSB repair (2-4). To repair a DSB, the DNA end is first processed to reveal a 3Ј single-stranded (ssDNA) tail, which is subsequently channeled into one of the recombinational pathways. The canonical gene conversion pathway is an evolutionarily conserved process carried out by the DNA strand exchange protein, Rad51 (5). Genes that are important for the gene conversion pathways are RAD51, RAD52, RAD54, RAD55, RAD57, and RFA1, which collectively define the RAD51-dependent recombination pathway (2, 4).In addition to the RAD51-dependent recombination, alternative repair pathways exist that are independent of RAD51 function. These pathways have different but overlapping genetic requirements from the classical gene conversion pathway (1, 6 -8). In S. cerevisiae, the major RAD51-independent pathway is single-stranded annealing (SSA). The SSA pathway does not involve Rad51-mediated invasion of dsDNA and the subsequent strand exchange steps. Instead, it occurs by annealing of complementary ssDNA on either side of the DSB, followed by removal of heterologous tails and ligation of the nicks (2). In addition to the recombination genes MRE11, RAD50, XRS2, RAD52, and RAD59 (6, 7), the SSA pathway also depends on the MSH2, MSH3 (7, 9), RAD1 and RAD10 (9, 10) genes. SSA in yeast is highly effective, accounting for 35-80% of DSB repair events when a DSB occurs between two direct repeats (2, 6, 11).The RAD52 gene encodes a 471-residue protein that is required for all homologous recombination events in S. cerevisiae (2, 4). The ϳ200-residue N terminus of Rad52 protein is highly conserved in most eukaryotic species; whereas the C terminus confers species-specific interaction...

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“…In our method, only the reporter gene is introduced in the first step. In instances where larger genomic regions are being edited, this feature reduces the size of the region that must be inserted initially by HDR, which increases efficiency, 30 and creates a stable genotype that can be used to eliminate the first stage CRISPR modification in any future experiments that modify the same locus.…”

Section: Discussionmentioning

confidence: 99%

Tools and strategies for scarless allele replacement inDrosophilausing CRISPR/Cas9

Lamb

Walker

Wittkopp

2016

Fly

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Genome editing via the CRISPR/Cas9 RNA-guided nuclease system has opened up exciting possibilities for genetic analysis. However, technical challenges associated with homology-directed repair have proven to be roadblocks for producing changes in the absence of unwanted, secondary mutations commonly known as "scars." To address these issues, we developed a 2-stage, markerassisted strategy to facilitate precise, "scarless" edits in Drosophila with a minimal requirement for molecular screening. Using this method, we modified 2 base pairs in a gene of interest without altering the final sequence of the CRISPR cut sites. We executed this 2-stage allele swap using a novel transformation marker that drives expression in the pupal wings, which can be screened for in the presence of common eye-expressing reporters. The tools we developed can be used to make a single change or a series of allelic substitutions in a region of interest in any D. melanogaster genetic background as well as in other Drosophila species.

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Factors Affecting Double-Strand Break-Induced Homologous Recombination in Mammalian Cells

Cited by 32 publications

References 31 publications

Microhomology-mediated and nonhomologous repair of a double-strand break in the chloroplast genome of Arabidopsis

Microhomology-mediated and nonhomologous repair of a double-strand break in the chloroplast genome of Arabidopsis

DNA Annealing Mediated by Rad52 and Rad59 Proteins

Tools and strategies for scarless allele replacement inDrosophilausing CRISPR/Cas9

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