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Genetic evidence that the non-homologous end-joining repair pathway is involved in LINE retrotransposition.

Suzuki J, Yamaguchi K, Kajikawa M, Ichiyanagi K, Adachi N, Koyama H, Takeda S, Okada N - PLoS Genet. (2009)

Bottom Line: LINEs mobilize via a process called retrotransposition.Deficiencies of NHEJ proteins decreased retrotransposition frequencies of both LINEs in these cells, suggesting that NHEJ is involved in LINE retrotransposition.More precise characterization of ZfL2-2 insertions in DT40 cells permitted us to consider the possibility of dual roles for NHEJ in LINE retrotransposition, namely to ensure efficient integration of LINEs and to restrict their full-length formation.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Midori-ku, Yokohama, Kanagawa, Japan.

ABSTRACT
Long interspersed elements (LINEs) are transposable elements that proliferate within eukaryotic genomes, having a large impact on eukaryotic genome evolution. LINEs mobilize via a process called retrotransposition. Although the role of the LINE-encoded protein(s) in retrotransposition has been extensively investigated, the participation of host-encoded factors in retrotransposition remains unclear. To address this issue, we examined retrotransposition frequencies of two structurally different LINEs--zebrafish ZfL2-2 and human L1--in knockout chicken DT40 cell lines deficient in genes involved in the non-homologous end-joining (NHEJ) repair of DNA and in human HeLa cells treated with a drug that inhibits NHEJ. Deficiencies of NHEJ proteins decreased retrotransposition frequencies of both LINEs in these cells, suggesting that NHEJ is involved in LINE retrotransposition. More precise characterization of ZfL2-2 insertions in DT40 cells permitted us to consider the possibility of dual roles for NHEJ in LINE retrotransposition, namely to ensure efficient integration of LINEs and to restrict their full-length formation.

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Retrotransposition assay in HeLa cells with NU7026.(A) Survival rate of HeLa cells treated with NU7026 in the presence or absence of etoposide. HeLa cells treated with these agents for 2 h were plated on a 100-mm plate. Three independent experiments were performed, and the means with standard deviations are shown. (B) The result of the retrotransposition assay in HeLa cells treated with NU7026. Retrotransposition frequency (RF) values are relative to those measured in the absence of NU7026. Two independent experiments were performed, and the means with standard deviations are shown.
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pgen-1000461-g004: Retrotransposition assay in HeLa cells with NU7026.(A) Survival rate of HeLa cells treated with NU7026 in the presence or absence of etoposide. HeLa cells treated with these agents for 2 h were plated on a 100-mm plate. Three independent experiments were performed, and the means with standard deviations are shown. (B) The result of the retrotransposition assay in HeLa cells treated with NU7026. Retrotransposition frequency (RF) values are relative to those measured in the absence of NU7026. Two independent experiments were performed, and the means with standard deviations are shown.

Mentions: To examine whether NHEJ is also involved in LINE retrotransposition in cells other than chicken DT40, we performed the retrotransposition assay in human HeLa cells (Figure 4). No knockout HeLa cell line is available, but DNA-PKcs kinase activity can be specifically inhibited by NU7026 [30]. We first confirmed that NU7026 kills HeLa cells in a dose-dependent manner only in the presence of a DSB inducer, etoposide [31]. HeLa cells treated with NU7026 became more sensitive to etoposide, indicating that the NHEJ repair capacity is suppressed by NU7026 (Figure 4A). The RFs of both ZfL2-2 and L1 decreased with increasing concentrations of NU7026, suggesting that the NHEJ pathway is also involved in LINE retrotransposition in HeLa cells (Figure 4B, Table S6, S7). Consistent with the results using DT40 cells, ZfL2-2 retrotransposition was more sensitive than L1 retrotransposition to NU7026.


Genetic evidence that the non-homologous end-joining repair pathway is involved in LINE retrotransposition.

Suzuki J, Yamaguchi K, Kajikawa M, Ichiyanagi K, Adachi N, Koyama H, Takeda S, Okada N - PLoS Genet. (2009)

Retrotransposition assay in HeLa cells with NU7026.(A) Survival rate of HeLa cells treated with NU7026 in the presence or absence of etoposide. HeLa cells treated with these agents for 2 h were plated on a 100-mm plate. Three independent experiments were performed, and the means with standard deviations are shown. (B) The result of the retrotransposition assay in HeLa cells treated with NU7026. Retrotransposition frequency (RF) values are relative to those measured in the absence of NU7026. Two independent experiments were performed, and the means with standard deviations are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2666801&req=5

pgen-1000461-g004: Retrotransposition assay in HeLa cells with NU7026.(A) Survival rate of HeLa cells treated with NU7026 in the presence or absence of etoposide. HeLa cells treated with these agents for 2 h were plated on a 100-mm plate. Three independent experiments were performed, and the means with standard deviations are shown. (B) The result of the retrotransposition assay in HeLa cells treated with NU7026. Retrotransposition frequency (RF) values are relative to those measured in the absence of NU7026. Two independent experiments were performed, and the means with standard deviations are shown.
Mentions: To examine whether NHEJ is also involved in LINE retrotransposition in cells other than chicken DT40, we performed the retrotransposition assay in human HeLa cells (Figure 4). No knockout HeLa cell line is available, but DNA-PKcs kinase activity can be specifically inhibited by NU7026 [30]. We first confirmed that NU7026 kills HeLa cells in a dose-dependent manner only in the presence of a DSB inducer, etoposide [31]. HeLa cells treated with NU7026 became more sensitive to etoposide, indicating that the NHEJ repair capacity is suppressed by NU7026 (Figure 4A). The RFs of both ZfL2-2 and L1 decreased with increasing concentrations of NU7026, suggesting that the NHEJ pathway is also involved in LINE retrotransposition in HeLa cells (Figure 4B, Table S6, S7). Consistent with the results using DT40 cells, ZfL2-2 retrotransposition was more sensitive than L1 retrotransposition to NU7026.

Bottom Line: LINEs mobilize via a process called retrotransposition.Deficiencies of NHEJ proteins decreased retrotransposition frequencies of both LINEs in these cells, suggesting that NHEJ is involved in LINE retrotransposition.More precise characterization of ZfL2-2 insertions in DT40 cells permitted us to consider the possibility of dual roles for NHEJ in LINE retrotransposition, namely to ensure efficient integration of LINEs and to restrict their full-length formation.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Midori-ku, Yokohama, Kanagawa, Japan.

ABSTRACT
Long interspersed elements (LINEs) are transposable elements that proliferate within eukaryotic genomes, having a large impact on eukaryotic genome evolution. LINEs mobilize via a process called retrotransposition. Although the role of the LINE-encoded protein(s) in retrotransposition has been extensively investigated, the participation of host-encoded factors in retrotransposition remains unclear. To address this issue, we examined retrotransposition frequencies of two structurally different LINEs--zebrafish ZfL2-2 and human L1--in knockout chicken DT40 cell lines deficient in genes involved in the non-homologous end-joining (NHEJ) repair of DNA and in human HeLa cells treated with a drug that inhibits NHEJ. Deficiencies of NHEJ proteins decreased retrotransposition frequencies of both LINEs in these cells, suggesting that NHEJ is involved in LINE retrotransposition. More precise characterization of ZfL2-2 insertions in DT40 cells permitted us to consider the possibility of dual roles for NHEJ in LINE retrotransposition, namely to ensure efficient integration of LINEs and to restrict their full-length formation.

Show MeSH
Related in: MedlinePlus