Low joining efficiency and non-conservative repair of two distant double-strand breaks in mouse embryonic stem cells

Boubakour-Azzouz, Imenne; Ricchetti, Miria

doi:10.1016/j.dnarep.2007.09.005

Cited by 10 publications

(10 citation statements)

References 43 publications

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“…Repair of the chromosome allows the growth of Spsensitive bacteria on Ara plates. The repair of two distant chromosomal DSBs is much less efficient than the repair of a single DSB also in NHEJ-proficient mammalian cells (frequency of 10 −2 -10 −6 and of approximately 10 −1 , respectively) (19,20,21,22). Nevertheless this observation, involving loss of an intervening sequence and formation of a new junction, clearly demonstrates the occurrence of end-joining.…”

mentioning

confidence: 63%

An end-joining repair mechanism in Escherichia coli

Chayot¹,

Montagne²,

Mazel

et al. 2010

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

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142

123

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Bridging broken DNA ends via nonhomologous end-joining (NHEJ) contributes to the evolution and stability of eukaryote genomes. Although some bacteria possess a simplified NHEJ mechanism, the human commensal Escherichia coli is thought to rely exclusively on homology-directed mechanisms to repair DNA double-strand breaks (DSBs). We show here that laboratory and pathogenic E. coli strains possess a distinct end-joining activity that repairs DSBs and generates genome rearrangements. This mechanism, named alternative end-joining (A-EJ), does not rely on the key NHEJ proteins Ku and Ligase-D which are absent in E. coli. Differently from classical NHEJ, A-EJ is characterized by extensive end-resection largely due to RecBCD, by overwhelming usage of microhomology and extremely rare DNA synthesis. We also show that A-EJ is dependent on the essential Ligase-A and independent on Ligase-B. Importantly, mutagenic repair requires a functional Ligase-A. Although generally mutagenic, accurate A-EJ also occurs and is frequent in some pathogenic bacteria. Furthermore, we show the acquisition of an antibiotic-resistance gene via A-EJ, refuting the notion that bacteria gain exogenous sequences only by recombination-dependent mechanisms. This finding demonstrates that E. coli can integrate unrelated, nonhomologous exogenous sequences by end-joining and it provides an alternative strategy for horizontal gene transfer in the bacterial genome. Thus, A-EJ contributes to bacterial genome evolution and adaptation to environmental challenges. Interestingly, the key features of A-EJ also appear in A-NHEJ, an alternative end-joining mechanism implicated in chromosomal translocations associated with human malignancies, and we propose that this mutagenic repair might have originated in bacteria.

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mentioning

confidence: 63%

An end-joining repair mechanism in Escherichia coli

Chayot¹,

Montagne²,

Mazel

et al. 2010

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

Self Cite

142

123

View full text Add to dashboard Cite

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“…Genetic studies seem to support this model as repair of a single DSB in mouse and human cells does not promote large scale genome rearrangements between heterologs, although they can be associated with regional loss of heterozygosity (LOH) and insertions with sequences of unknown origin [20,31–33,81]. Similarly, multiple DSBs on the same chromosome do not significantly promote large-scale genome rearrangements in mouse or human cell lines, although the efficiency of repair decreases as the distance between two DSBs increases (up to 9 kb apart) [82,83]. …”

Section: Discussionmentioning

confidence: 99%

Double-Strand Break Repair by Interchromosomal Recombination: An In Vivo Repair Mechanism Utilized by Multiple Somatic Tissues in Mammals

et al. 2013

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Homologous recombination (HR) is essential for accurate genome duplication and maintenance of genome stability. In eukaryotes, chromosomal double strand breaks (DSBs) are central to HR during specialized developmental programs of meiosis and antigen receptor gene rearrangements, and form at unusual DNA structures and stalled replication forks. DSBs also result from exposure to ionizing radiation, reactive oxygen species, some anti-cancer agents, or inhibitors of topoisomerase II. Literature predicts that repair of such breaks normally will occur by non-homologous end-joining (in G1), intrachromosomal HR (all phases), or sister chromatid HR (in S/G2). However, no in vivo model is in place to directly determine the potential for DSB repair in somatic cells of mammals to occur by HR between repeated sequences on heterologs (i.e., interchromosomal HR). To test this, we developed a mouse model with three transgenes—two nonfunctional green fluorescent protein (GFP) transgenes each containing a recognition site for the I-SceI endonuclease, and a tetracycline-inducible I-SceI endonuclease transgene. If interchromosomal HR can be utilized for DSB repair in somatic cells, then I-SceI expression and induction of DSBs within the GFP reporters may result in a functional GFP+ gene. Strikingly, GFP+ recombinant cells were observed in multiple organs with highest numbers in thymus, kidney, and lung. Additionally, bone marrow cultures demonstrated interchromosomal HR within multiple hematopoietic subpopulations including multi-lineage colony forming unit–granulocyte-erythrocyte-monocyte-megakaryocte (CFU-GEMM) colonies. This is a direct demonstration that somatic cells in vivo search genome-wide for homologous sequences suitable for DSB repair, and this type of repair can occur within early developmental populations capable of multi-lineage differentiation.

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“…On the other hand, absence of Ku70, a member of the NHEJ pathway, shows no obvious increase in chromosome fragmentation in ES cells [50]. Further support derives from the observation that ES cells are unable to efficiently repair an integrated reporter plasmid into which 2 double-strand cuts had been introduced about 8.8 kb apart [51]. The reporter had no regions of homology around the break sites requiring utilization of NHEJ for repair rather than HRR.…”

Section: Discussionmentioning

confidence: 99%

Mouse Embryonic Stem Cells, but Not Somatic Cells, Predominantly Use Homologous Recombination to Repair Double-Strand DNA Breaks

Tichy

Pillai

Deng

et al. 2010

Stem Cells and Development

143

151

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Embryonic stem (ES) cells give rise to all cell types of an organism. Since mutations at this embryonic stage would affect all cells and be detrimental to the overall health of an organism, robust mechanisms must exist to ensure that genomic integrity is maintained. To test this proposition, we compared the capacity of murine ES cells to repair DNA double-strand breaks with that of differentiated cells. Of the 2 major pathways that repair double-strand breaks, error-prone nonhomologous end joining (NHEJ) predominated in mouse embryonic fibroblasts, whereas the high fidelity homologous recombinational repair (HRR) predominated in ES cells. Microhomology-mediated end joining, an emerging repair pathway, persisted at low levels in all cell types examined. The levels of proteins involved in HRR and microhomology-mediated end joining were highly elevated in ES cells compared with mouse embryonic fibroblasts, whereas those for NHEJ were quite variable, with DNA Ligase IV expression low in ES cells. The half-life of DNA Ligase IV protein was also low in ES cells. Attempts to increase the abundance of DNA Ligase IV protein by overexpression or inhibition of its degradation, and thereby elevate NHEJ in ES cells, were unsuccessful. When ES cells were induced to differentiate, however, the level of DNA Ligase IV protein increased, as did the capacity to repair by NHEJ. The data suggest that preferential use of HRR rather than NHEJ may lend ES cells an additional layer of genomic protection and that the limited levels of DNA Ligase IV may account for the low level of NHEJ activity.

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Low joining efficiency and non-conservative repair of two distant double-strand breaks in mouse embryonic stem cells

Cited by 10 publications

References 43 publications

An end-joining repair mechanism in Escherichia coli

An end-joining repair mechanism in Escherichia coli

Double-Strand Break Repair by Interchromosomal Recombination: An In Vivo Repair Mechanism Utilized by Multiple Somatic Tissues in Mammals

Mouse Embryonic Stem Cells, but Not Somatic Cells, Predominantly Use Homologous Recombination to Repair Double-Strand DNA Breaks

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