Integration of exogenous DNA into mouse embryonic stem cell chromosomes shows preference into genes and frequent modification at junctions

Suzuki, Keiichiro; Ohbayashi, Fumi; Nikaido, Itoshi; Okuda, Akihiko; Takaki, Haruyoshi; Okazaki, Yasushi; Mitani, Kohnosuke

doi:10.1007/s10577-010-9111-5

Cited by 5 publications

(6 citation statements)

References 33 publications

(49 reference statements)

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“…However, a more likely possibility for LIG4-independent integration may be a synthesis-dependent microhomology-mediated end-joining (SD-MMEJ) mechanism, as proposed by Yu and McVey [22]. This model well explains frequent terminal modifications observed at plasmid-genome junctions in random integrants from mouse ES cells [23] as well as little or no apparent junctional microhomologies in LIG4 -null cells ([21]; our unpublished observations).…”

Section: Resultsmentioning

confidence: 52%

“…Moreover, SINEs tend to distribute in gene-rich GC-rich regions, while LINEs in gene-poor AT-rich regions [30]. In this regard, recent work has reported that integration of exogenous DNA into mouse ES cell chromosomes shows preference into genes [23]. Alternatively or additionally, as LINEs present in the targeting vectors are short fragments (<300 bp)(Figure S1 in File S1), these LINE fragments may lack the ability to facilitate random integration.…”

Section: Resultsmentioning

confidence: 99%

“…Importantly, for SSA or microhomology-mediated alternative end-joining to bring about random integration, not only must the end of the vector be extensively deleted, but also the target genome should have a pre-existing DSB at or near a SINE/LINE fragment. This is likely enough, given the observed frequent terminal deletions of transfected plasmid [23] and the high abundance of SINE/LINE fragments in the genome. However, a more likely possibility for LIG4-independent integration may be a synthesis-dependent microhomology-mediated end-joining (SD-MMEJ) mechanism, as proposed by Yu and McVey [22].…”

Section: Resultsmentioning

confidence: 99%

“…[23] have reported that chromosomal integration of plasmid DNA (a non-targeting vector) transfected into mouse embryonic stem (ES) cells is mostly a complex reaction with frequent terminal deletions of the plasmid and the genome. Interestingly, sequence analysis of those random integrants revealed a frequent use of microhomologies at the plasmid-genome junctions [23]. This finding may suggest the occurrence of random integration events via alternative end-joining, given the aforementioned microhomology preference of this mechanism in repair of DSBs.…”

Section: Introductionmentioning

confidence: 99%

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Analysis of the Role of Homology Arms in Gene-Targeting Vectors in Human Cells

et al. 2014

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Random integration of targeting vectors into the genome is the primary obstacle in human somatic cell gene targeting. Non-homologous end-joining (NHEJ), a major pathway for repairing DNA double-strand breaks, is thought to be responsible for most random integration events; however, absence of DNA ligase IV (LIG4), the critical NHEJ ligase, does not significantly reduce random integration frequency of targeting vector in human cells, indicating robust integration events occurring via a LIG4-independent mechanism. To gain insights into the mechanism and robustness of LIG4-independent random integration, we employed various types of targeting vectors to examine their integration frequencies in LIG4-proficient and deficient human cell lines. We find that the integration frequency of targeting vector correlates well with the length of homology arms and with the amount of repetitive DNA sequences, especially SINEs, present in the arms. This correlation was prominent in LIG4-deficient cells, but was also seen in LIG4-proficient cells, thus providing evidence that LIG4-independent random integration occurs frequently even when NHEJ is functionally normal. Our results collectively suggest that random integration frequency of conventional targeting vectors is substantially influenced by homology arms, which typically harbor repetitive DNA sequences that serve to facilitate LIG4-independent random integration in human cells, regardless of the presence or absence of functional NHEJ.

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Section: Resultsmentioning

confidence: 52%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Analysis of the Role of Homology Arms in Gene-Targeting Vectors in Human Cells

et al. 2014

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“…1b). Consistent with the intrinsic complexity of RI events38, the precise mechanisms for several junctions associated with templated insertions were difficult to interpret (Supplementary Data 1), but some junctions showed a simple pattern of templated insertions (that is, ≥6-bp direct or inverted repeats), including those copied sequences from the other side of the vector-genome junctions (Fig. 1e).…”

Section: Resultsmentioning

confidence: 94%

Dual loss of human POLQ and LIG4 abolishes random integration

2017

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Homologous recombination-mediated gene targeting has greatly contributed to genetic analysis in a wide range of species, but is highly inefficient in human cells because of overwhelmingly frequent random integration events, whose molecular mechanism remains elusive. Here we show that DNA polymerase θ, despite its minor role in chromosomal DNA repair, substantially contributes to random integration, and that cells lacking both DNA polymerase θ and DNA ligase IV, which is essential for non-homologous end joining (NHEJ), exhibit 100% efficiency of spontaneous gene targeting by virtue of undetectable levels of random integration. Thus, DNA polymerase θ-mediated end joining is the sole homology-independent repair route in the absence of NHEJ and, intriguingly, their combined absence reveals rare Alu-Alu recombination events utilizing a stretch of homology. Our findings provide new insights into the mechanics of foreign DNA integration and the role of DNA polymerase θ in human genome maintenance.

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