Alternative end-joining pathway(s): Bricolage at DNA breaks

Frit, Philippe; Barboule, Nadia; Yuan, Ying; Gómez, Dennis; Calsou, Patrick

doi:10.1016/j.dnarep.2014.02.007

Cited by 126 publications

(121 citation statements)

References 310 publications

(493 reference statements)

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“…S14D). These findings indicate that NHEJ and A-EJ, but not HR, are the most prevalent pathways of DNA repair for lesions occurring at nuclear membrane-associated chromatin and reveal for the first time that A-EJ takes place as a main pathway and not as a backup pathway activated solely in instances where there is a DNA repair factor deficiency (Frit et al 2014).…”

Section: Resultsmentioning

confidence: 91%

Nuclear position dictates DNA repair pathway choice

et al. 2014

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Faithful DNA repair is essential to avoid chromosomal rearrangements and promote genome integrity. Nuclear organization has emerged as a key parameter in the formation of chromosomal translocations, yet little is known as to whether DNA repair can efficiently occur throughout the nucleus and whether it is affected by the location of the lesion. Here, we induce DNA double-strand breaks (DSBs) at different nuclear compartments and follow their fate. We demonstrate that DSBs induced at the nuclear membrane (but not at nuclear pores or nuclear interior) fail to rapidly activate the DNA damage response (DDR) and repair by homologous recombination (HR). Real-time and superresolution imaging reveal that DNA DSBs within lamina-associated domains do not migrate to more permissive environments for HR, like the nuclear pores or the nuclear interior, but instead are repaired in situ by alternative end-joining. Our results are consistent with a model in which nuclear position dictates the choice of DNA repair pathway, thus revealing a new level of regulation in DSB repair controlled by spatial organization of DNA within the nucleus.

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Section: Resultsmentioning

confidence: 91%

Nuclear position dictates DNA repair pathway choice

et al. 2014

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“…2a, 2b). Whether the residual NHEJ activity relies on the KU/Ligase IV independent alternative end-joining mechanism 15 remains to be determined.…”

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confidence: 99%

Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells

et al. 2015

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The insertion of precise genetic modifications by genome editing tools such as CRISPR-Cas9 is limited by the relatively low efficiency of homology-directed repair (HDR) compared with the higher efficiency of the non-homologous end joining (NHEJ) pathway. To enhance HDR, enabling the insertion of precise genetic modifications, we suppressed the NHEJ key molecules KU70, KU80 or DNA Ligase IV by gene silencing, the Ligase IV inhibitor SCR7 or the coexpression of adenovirus 4 E1B55K and E4orf6 proteins in a `traffic light´ and other reporter systems. Suppression of KU70 and DNA Ligase IV promotes the efficiency of HDR 4-5-fold. When co-expressed with the Cas9 system, E1B55K and E4orf6 improved the efficiency of HDR up to 8-fold and essentially abolished NHEJ activity in both human and mouse cell lines. Our findings provide useful tools to improve the frequency of precise gene modifications in mammalian cells.The clustered, regularly interspaced, short palindromic repeats (CRISPRs)-Cas9 endonuclease represent a versatile tool for genome engineering, enabling the induction of site-specific genomic double-strand breaks (DSBs) by single guide RNAs (sgRNAs) 1 .In mammalian cells DSBs are mostly repaired by the non-homologous end joining (NHEJ) pathway 2,3 , frequently leading to the loss of nucleotides from the DSB ends. This enables the efficient construction of knockout alleles through the induction of frameshift To quantitatively determine the outcome of CRISPR-induced DSB repair, we first generated human HEK293 cells with a `traffic light´ reporter 7 (TLR) vector integrated into the AAVS1 locus 8 (Fig. 1a). HEK293 cells were transfected with an AAVS1 targeting vector carrying the TLR insert and Cas9 and AAVS1-specific sgRNA expression plasmids ( Supplementary Fig. 1 , and up to 7-fold by the Ad4 protein pair ( Fig. 2c and Supplementary Fig. 2.2).Titration of SCR7 on AAVS1 TLR/+ cells showed an optimal effect at 1 µM concentration ( Supplementary Fig. 2.3). In the presence of two shRNAs, SCR7 or Ad4 proteins we noticed diminished fluorescence signals within the population of Venus + cells at 72h after transfection (Fig. 2a, Supplementary Fig. 2.1, 2.3c), indicating reduced Venus expression in cells undergoing NHEJ blockade, possibly caused by local chromatin remodeling through an extended DNA damage response 13,14 . However, Venus expression was normal in clones established from AAVS1 TLR/+ cells targeted in the presence of Ad4 proteins, indicating that this effect is only transient (Supplementary Fig. 2.3d). From the sample expressing the Ad4 proteins, Venus + cells were sorted, and we established 24 clones to confirm the integrity of the repaired TLR loci using PCR and sequence analysis (Supplementary Table 2). In contrast to the increase of Venus + cells, RFP + cells decreased from 3% in the controls to 1.7%, 1.4% or 0.6% in the presence of 5 shRNAs, SCR7 or Ad4 proteins, respectively (Fig. 2a, 2b). Whether the residual NHEJ activity relies on the KU/Ligase IV independent alternative end-joining mechanism 15 rem...

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“…The results suggested the existence of multiple DNA ligase-containing complexes in A-EJ. sometimes are controversial (5,20,21). For example, whether A-EJ is a completely independent new pathway or an alternative c-NHEJ pathway in which alternative components could substitute the missing c-NHEJ factors is still debatable.…”

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confidence: 99%

Ligase I and ligase III mediate the DNA double-strand break ligation in alternative end-joining

Duan

Shu

et al. 2016

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

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In eukaryotes, DNA double-strand breaks (DSBs), one of the most harmful types of DNA damage, are repaired by homologous repair (HR) and nonhomologous end-joining (NHEJ). Surprisingly, in cells deficient for core classic NHEJ factors such as DNA ligase IV (Lig4), substantial end-joining activities have been observed in various situations, suggesting the existence of alternative end-joining (A-EJ) activities. Several putative A-EJ factors have been proposed, although results are mostly controversial. By using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, we generated mouse CH12F3 cell lines in which, in addition to Lig4, either Lig1 or nuclear Lig3, representing the cells containing a single DNA ligase (Lig3 or Lig1, respectively) in their nucleus, was completely ablated. Surprisingly, we found that both Lig1-and Lig3-containing complexes could efficiently catalyze A-EJ for class switching recombination (CSR) in the IgH locus and chromosomal deletions between DSBs generated by CRISPR/Cas9 in cis-chromosomes. However, only deletion of nuclear Lig3, but not Lig1, could significantly reduce the interchromosomal translocations in Lig4 M ammalian genomes are subjected substantial DNA damage from both endogenous processes [e.g., DNA replication, class switching recombination (CSR), etc.] and exogenous resources (e.g., ionic radiation, DNA-damaging chemicals, etc.). Evolutionarily conserved DNA repair pathways are essential to maintain both the structure integrity and the information accuracy of the genome (1). In eukaryotes, DNA double-strand breaks (DSBs), one of the most dangerous and severe types of DNA damage, are repaired mainly by two evolutionarily conserved repair pathways: homologous repair (HR) and nonhomologous end-joining (NHEJ) (2). NHEJ directly ligates two broken DSB ends, whereas HR uses a homologous template (in most of cases, the sister chromosome) for repair. Although NHEJ is simpler and faster than HR, repair by NHEJ often leads to change of sequences in the repair junctions via deletion, insertion, and mutations. Most importantly, NHEJ can also directly ligate two distant DSBs (both in cis-and transchromosomes), therefore leading to chromosomal deletions and translocations that are tightly linked to the genome evolution and carcinogenesis (3).The NHEJ in eukaryotes has been extensively studied in the last two decades (4-6). Mechanistically, NHEJ could be separated into several steps. First, in the DSB binding and tethering step, the DSB ends are recognized and bound by Ku70/Ku80 heterodimers that function as docking sites for other NHEJ factors. In the next end-processing step, nuclease and DNA polymerase activities are recruited to remove damaged or mismatched nucleotides and prepare the broken ends for ligation. Finally, DNA ligase IV (Lig4) complex, consisting of DNA Lig4 and its cofactor X-ray repair cross-complementing protein 4 (XRCC4), as well as the newly identified XRCC4-interacting factor (XLF), reseals the DSBs. Both V(D)J ...

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Alternative end-joining pathway(s): Bricolage at DNA breaks

Cited by 126 publications

References 310 publications

Nuclear position dictates DNA repair pathway choice

Nuclear position dictates DNA repair pathway choice

Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells

Ligase I and ligase III mediate the DNA double-strand break ligation in alternative end-joining

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