T-DNA integration in plants results from polymerase-θ-mediated DNA repair

Kregten, Maartje van; Pater, Sylvia de; Romeijn, Ron J.; Schendel, Robin van; Hooykaas, Paul J. J.; Tijsterman, Marcel

doi:10.1038/nplants.2016.164

Cited by 125 publications

(146 citation statements)

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“…The ku80 and ku80 parp1 parp2 mutant lines appeared to have more templated-insertion events than wild-type and parp1 parp2 lines, although such insertions were found in all four genotypes (Table S3). Therefore, the templated insertions probably resulted from a KU80- and PARPs-independent alternative end-joining mechanism, such as that mediated by the recently discovered Pol θ (van Kregten et al 2016). …”

Section: Resultsmentioning

confidence: 99%

“…In plants, templated insertions were also observed after DSB-induced repair in Arabidopsis and tobacco (Shirley et al 1992; Gorbunova and Levy 1997; Salomon and Puchta 1998; Lloyd et al 2012; Vu et al 2014). Furthermore, a recent study showed that the Arabidopsis Pol θ ortholog Tebichi ( Teb ) is essential for T-DNA integration (van Kregten et al 2016). Templated insertions were found at the repair junctions of T-DNA inserts, and it was shown that teb mutants were resistant to T-DNA integration and very sensitive to the DNA-damaging agents bleomycin and MMS.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

CRISPR/Cas9-Induced Double-Strand Break Repair in Arabidopsis Nonhomologous End-Joining Mutants

Shen¹,

Strunks²,

Klemann³

et al. 2017

G3 Genes|Genomes|Genetics

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Double-strand breaks (DSBs) are one of the most harmful DNA lesions. Cells utilize two main pathways for DSB repair: homologous recombination (HR) and nonhomologous end-joining (NHEJ). NHEJ can be subdivided into the KU-dependent classical NHEJ (c-NHEJ) and the more error-prone KU-independent backup-NHEJ (b-NHEJ) pathways, involving the poly (ADP-ribose) polymerases (PARPs). However, in the absence of these factors, cells still seem able to adequately maintain genome integrity, suggesting the presence of other b-NHEJ repair factors or pathways independent from KU and PARPs. The outcome of DSB repair by NHEJ pathways can be investigated by using artificial sequence-specific nucleases such as CRISPR/Cas9 to induce DSBs at a target of interest. Here, we used CRISPR/Cas9 for DSB induction at the Arabidopsis cruciferin 3 (CRU3) and protoporphyrinogen oxidase (PPO) genes. DSB repair outcomes via NHEJ were analyzed using footprint analysis in wild-type plants and plants deficient in key factors of c-NHEJ (ku80), b-NHEJ (parp1 parp2), or both (ku80 parp1 parp2). We found that larger deletions of >20 bp predominated after DSB repair in ku80 and ku80 parp1 parp2 mutants, corroborating with a role of KU in preventing DSB end resection. Deletion lengths did not significantly differ between ku80 and ku80 parp1 parp2 mutants, suggesting that a KU- and PARP-independent b-NHEJ mechanism becomes active in these mutants. Furthermore, microhomologies and templated insertions were observed at the repair junctions in the wild type and all mutants. Since these characteristics are hallmarks of polymerase θ-mediated DSB repair, we suggest a possible role for this recently discovered polymerase in DSB repair in plants.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9-Induced Double-Strand Break Repair in Arabidopsis Nonhomologous End-Joining Mutants

Shen¹,

Strunks²,

Klemann³

et al. 2017

G3 Genes|Genomes|Genetics

Self Cite

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show abstract

“…A recent breakthrough showed that T-DNA integration in A. thaliana requires the polymerase theta (Pol θ, At4g32700) (van Kregten et al, 2016). This discovery was inspired by the similar signature of doublestrand breaks (DSBs) of T-DNA integration with other systems.…”

Section: Integration Into the Plant Genome And Expression Of The T-dnamentioning

confidence: 99%

“…Based on the knowledge of molecular action of Pol θ, it has been proposed that T-DNA could target the random DSBs in the genome and the microhomology between the ends of T-DNA and the DSBs is required for the integration of T-DNA. Enhanced understanding of the mechanism of T-DNA integration may allow researchers to improve transgene targeting by introducing artificial breaks in the plant genome, which ideally will lead to specific integration of DNA fragments in a designated location (van Kregten et al, 2016).…”

Section: Integration Into the Plant Genome And Expression Of The T-dnamentioning

confidence: 99%

Agrobacterium-Mediated Plant Transformation: Biology and Applications

Hwang

Lai

2017

The Arabidopsis Book

230

142

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“…In addition, Pol θ plays a critical role in the generation of insertions during alternative end joining repair of collapsed replication forks in C . elegans [19, 20, 26] and during T-DNA insertion in Arabidopsis [27]. …”

Section: Introductionmentioning

confidence: 99%

Drosophila DNA polymerase theta utilizes both helicase-like and polymerase domains during microhomology-mediated end joining and interstrand crosslink repair

et al. 2017

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Double strand breaks (DSBs) and interstrand crosslinks (ICLs) are toxic DNA lesions that can be repaired through multiple pathways, some of which involve shared proteins. One of these proteins, DNA Polymerase θ (Pol θ), coordinates a mutagenic DSB repair pathway named microhomology-mediated end joining (MMEJ) and is also a critical component for bypass or repair of ICLs in several organisms. Pol θ contains both polymerase and helicase-like domains that are tethered by an unstructured central region. While the role of the polymerase domain in promoting MMEJ has been studied extensively both in vitro and in vivo, a function for the helicase-like domain, which possesses DNA-dependent ATPase activity, remains unclear. Here, we utilize genetic and biochemical analyses to examine the roles of the helicase-like and polymerase domains of Drosophila Pol θ. We demonstrate an absolute requirement for both polymerase and ATPase activities during ICL repair in vivo. However, similar to mammalian systems, polymerase activity, but not ATPase activity, is required for ionizing radiation-induced DSB repair. Using a site-specific break repair assay, we show that overall end-joining efficiency is not affected in ATPase-dead mutants, but there is a significant decrease in templated insertion events. In vitro, Pol θ can efficiently bypass a model unhooked nitrogen mustard crosslink and promote DNA synthesis following microhomology annealing, although ATPase activity is not required for these functions. Together, our data illustrate the functional importance of the helicase-like domain of Pol θ and suggest that its tethering to the polymerase domain is important for its multiple functions in DNA repair and damage tolerance.

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T-DNA integration in plants results from polymerase-θ-mediated DNA repair

Cited by 125 publications

References 37 publications

CRISPR/Cas9-Induced Double-Strand Break Repair in Arabidopsis Nonhomologous End-Joining Mutants

CRISPR/Cas9-Induced Double-Strand Break Repair in Arabidopsis Nonhomologous End-Joining Mutants

Agrobacterium-Mediated Plant Transformation: Biology and Applications

Drosophila DNA polymerase theta utilizes both helicase-like and polymerase domains during microhomology-mediated end joining and interstrand crosslink repair

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