Impact of non-homologous end-joining deficiency on random and targeted DNA integration: implications for gene targeting

Iiizumi, Susumu; Kurosawa, Aya; So, Sairei; Ishii, Yasuyuki; Chikaraishi, Yoshito; Ishii, Ayako; Koyama, Hideki; Adachi, Noritaka

doi:10.1093/nar/gkn649

Cited by 53 publications

(66 citation statements)

References 60 publications

(65 reference statements)

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“…These findings clearly indicate that NHEJ is not the sole mechanism of random integration in human somatic cells, and suggest the contribution of alternative end-joining to the residual random integration events by non-homologous recombination. Intriguingly, unlike vectors with no or shorter homology arms, integration frequency of targeting vectors with long homology arms was not affected by LIG4 deficiency [40] (Figure 1B, C; data not shown). It could be that in the absence of NHEJ, homology arms of the targeting vector served to prevent marker gene loss caused by large deletion (chew-back); however, as these homology arms contain a number of Alu elements, it is more likely that homology arms serve to trigger random integration in an NHEJ-independent fashion.…”

Section: Impact Of Dsb Repair Deficiency On Targeted and Random Integmentioning

confidence: 86%

“…In human somatic cells, however, suppression of NHEJ does not result in decreased random-integration frequency, although the efficiency of gene targeting can be increased [40] (Figure 1B-D). These findings clearly indicate that NHEJ is not the sole mechanism of random integration in human somatic cells, and suggest the contribution of alternative end-joining to the residual random integration events by non-homologous recombination.…”

Section: Impact Of Dsb Repair Deficiency On Targeted and Random Integmentioning

confidence: 98%

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Repair of Accidental DNA Double-Strand Breaks in the Human Genome and Its Relevance to Vector DNA Integration

Adachi

Saito²,

Kurosawa³

2014

Gene Technology

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Efficient repair of chromosomal DNA damage is crucial for cells to maintain genome integrity. DNA double-strand breaks (DSBs) are the most severe type of DNA lesions that can be caused by various exogenous and endogenous mechanisms, such as ionizing radiation, reactive oxygen species, topoisomerase poisons, or replication errors [1]. DSBs, if left unrepaired or mis-repaired, lead to cell death or chromosomal aberrations [2,3]. Human cells have evolved two fundamentally different mechanisms for repairing chromosomal DSBs, homologous recombination (HR) and non-homologous end-joining (NHEJ) [4]. NHEJ not only repairs accidental (non-physiological) DSBs, but is also essential for rejoining physiological DSBs that arise in the process of V(D)J recombination in B and T lymphocytes and class switch recombination in mature B cells [3].A wide variety of proteins have been identified thus far that contribute to the HR and NHEJ machineries [5]. HR is a highly complicated process of DNA transaction, in which Rad51 protein plays an essential role in DNA strand exchange with the aid of several other proteins such as Rad54, Brca2, Rad52, and Rad51 paralogs [6,7]. For HR to occur, DSBs should be processed (i.e., end-resected) to produce a long 3'-overhang single-stranded DNA [8,9], and recent studies have identified a number of proteins involved in end resection or its regulation; among these, Mre11 and CtIP play essential roles in the initial step of end resection [9][10][11]. In contrast to HR, NHEJ is thought to be a rather simpler process that requires, at least biochemically, only four proteins (two protein complexes); specifically, Ku, a heterodimer of Ku70 and Ku80, initiates an NHEJ reaction by binding to the ends of a DSB, and the DNA ligase complex composed of Xrcc4 and Ligase IV (Lig4) seals the ends to complete repair [3]. In most cases, however, many other proteins do participate in NHEJ-mediated repair to trim the DSB ends, which are typically non-ligatable or non-compatible. These additional NHEJ factors involve DNA-PKcs, Artemis, XLF, and DNA polymerase µ/λ; DNA-PKcs and Artemis have evolved in higher eukaryotes and do not exist in yeasts [3,12]. In addition to the classical pathway of NHEJ, recent evidence indicates the existence of a more error-prone mechanism of NHEJ called alternative endjoining that plays a role in DSB repair [3,13]. Alternative end-joining is Ku/Lig4 independent and the precise mechanism remains largely unclear, although PARP1, Ligase III, and several factors involved in end resection (to initiate HR) have been implicated in DSB repair via alternative end-joining [14][15][16].Which DSB repair pathway is beneficial for cells to preserve genome integrity? NHEJ (the classical NHEJ pathway) repairs broken DNA ends with little or no homology and is often associated with nucleotide loss, whereas HR allows for accurate repair of DSBs with the use of homologous DNA sequence, usually located on a sister chromatid [3,4,12]. Such difference in accuracy between the two pathways, however, does not mean...

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Section: Impact Of Dsb Repair Deficiency On Targeted and Random Integmentioning

confidence: 86%

Section: Impact Of Dsb Repair Deficiency On Targeted and Random Integmentioning

confidence: 98%

Repair of Accidental DNA Double-Strand Breaks in the Human Genome and Its Relevance to Vector DNA Integration

Adachi

Saito²,

Kurosawa³

2014

Gene Technology

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“…For example, iPS cells have been shown to be more stably maintained under low oxygen culture conditions ( [37] and our unpublished observations). Even more importantly, as random integration is a major obstacle in gene targeting via homologous recombination, a decreased random integration frequency should be preferable to increasing the frequency of targeted gene inactivation/ correction [7]. It is therefore expected that artificial manipulation of random integration will contribute to significant improvements in gene-targeting technology.…”

Section: Discussionmentioning

confidence: 99%

“…Random integration assays were carried out using Nalm-6 cells, essentially as described [7,19]. Briefly, 4×10…”

Section: Transfection and Integration Assaysmentioning

confidence: 99%

“…A practical application of nonhomologous recombination is the generation of transfectants (i.e., random integrants) that stably express a transgene(s) of interest. Although the precise mechanism of random integration is not fully understood, it is believed that random integration results from non-homologous recombination-mediated repair, particularly NHEJ, of a spontaneous chromosomal DSB accidentally induced by endogenous factors [5][6][7]. In this study, we focused on DNA topoisomerase II (Top2) and reactive oxygen species (ROS) as the endogenous factors that induce DSBs causative of random integration.…”

Section: Introductionmentioning

confidence: 99%

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Endogenous Factors Causative of Spontaneous DNA Damage that Leads to Random Integration in Human Cells

Kamekawa

Kurosawa²,

Umehara³

et al. 2013

Gene Technology

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Random integration is a phenomenon in which transfected DNA molecules integrate into (random sites of) the host genome via non-homologous recombination. Although it is assumed that repair of DNA double-strand breaks leads to random integration events, how these endogenous DNA lesions are generated in living cells is poorly understood. In this study, we present evidence that DNA topoisomerase IIα (Top2α) and reactive oxygen species (ROS) are responsible for causing genomic DNA damage that leads to random integration. Specifically, we employed a human pre-B lymphocyte cell line to examine the effects of cellular Top2 expression levels and oxygen concentrations during cell culture. We find that treating cells with Top2α siRNA significantly reduces random integration frequency, while the absence of Top2β had little or no impact. We also show that cells continuously cultured under low (3%) oxygen culture conditions after electroporation display reduced random integration frequency compared to that under normal (21%) oxygen conditions. These findings support the notion that Top2α protein and ROS are endogenous factors that can produce DNA damage leading to random integration of transfected DNA in human cells.

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Ethical and Safety Issues

Prazeres¹

2011

Plasmid Biopharmaceuticals

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Impact of non-homologous end-joining deficiency on random and targeted DNA integration: implications for gene targeting

Cited by 53 publications

References 60 publications

Repair of Accidental DNA Double-Strand Breaks in the Human Genome and Its Relevance to Vector DNA Integration

Repair of Accidental DNA Double-Strand Breaks in the Human Genome and Its Relevance to Vector DNA Integration

Endogenous Factors Causative of Spontaneous DNA Damage that Leads to Random Integration in Human Cells

Ethical and Safety Issues

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