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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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Characterization of ZfL2-2 insertions in DT40 cells.Abbreviations are as defined for Figure 1. (A) ZfL2-2 insertions isolated from DT40 cells. The top diagram shows the full-length ZfL2-2 element containing the mneoI400/ColE1 cassette (Cassette) in the 3′ UTR. ORF, open reading frame. Each horizontal line represents one of the 26, 25, 24 or 27 ZfL2-2 insertions isolated from the various DT40 cell lines. Blue lines represent insertions with a 5′ truncation. Red lines represent full-length insertions. The dashed line in Art−/− indicates a deletion. (B) A box-and-whisker plot shows the median (red line), the first and third quartiles, and the upper and lower limits of the length of insertions indicated in (A). P values less than 0.05 are indicated (Mann-Whitney U test). (C) Full-length vs. truncated elements. The ZfL2-2 insertions in (A) were categorized by the absence (Full) or presence (Truncated) of a 5′ truncation. The number of insertions identified is indicated inside each bar. P values less than 0.05 are indicated (two-sided Fisher's exact test). (D) Target site alterations. The ZfL2-2 insertions shown in (A) were categorized with regard to target site alterations. The number of insertions identified is indicated inside each bar. L-TST, long target site truncation (>20 bp). S-TST, short target site truncation (≤20 bp). BEJ, blunt end joining. S-TSD, short target site duplication (≤20 bp). L-TSD, long target site duplication (>20 bp). (E) Short target site alterations. The ZfL2-2 insertions with short target site alterations in (D) were compared. The number of insertions identified is indicated inside each bar. Abbreviations and definitions are as for panel D. P values less than 0.05 are indicated (Wilcoxon Rank Sum test).
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pgen-1000461-g003: Characterization of ZfL2-2 insertions in DT40 cells.Abbreviations are as defined for Figure 1. (A) ZfL2-2 insertions isolated from DT40 cells. The top diagram shows the full-length ZfL2-2 element containing the mneoI400/ColE1 cassette (Cassette) in the 3′ UTR. ORF, open reading frame. Each horizontal line represents one of the 26, 25, 24 or 27 ZfL2-2 insertions isolated from the various DT40 cell lines. Blue lines represent insertions with a 5′ truncation. Red lines represent full-length insertions. The dashed line in Art−/− indicates a deletion. (B) A box-and-whisker plot shows the median (red line), the first and third quartiles, and the upper and lower limits of the length of insertions indicated in (A). P values less than 0.05 are indicated (Mann-Whitney U test). (C) Full-length vs. truncated elements. The ZfL2-2 insertions in (A) were categorized by the absence (Full) or presence (Truncated) of a 5′ truncation. The number of insertions identified is indicated inside each bar. P values less than 0.05 are indicated (two-sided Fisher's exact test). (D) Target site alterations. The ZfL2-2 insertions shown in (A) were categorized with regard to target site alterations. The number of insertions identified is indicated inside each bar. L-TST, long target site truncation (>20 bp). S-TST, short target site truncation (≤20 bp). BEJ, blunt end joining. S-TSD, short target site duplication (≤20 bp). L-TSD, long target site duplication (>20 bp). (E) Short target site alterations. The ZfL2-2 insertions with short target site alterations in (D) were compared. The number of insertions identified is indicated inside each bar. Abbreviations and definitions are as for panel D. P values less than 0.05 are indicated (Wilcoxon Rank Sum test).

Mentions: To determine in which step of the retrotransposition reaction each NHEJ factor is involved, we determined and analyzed the 5′ and 3′ junction sequences of 102 ZfL2-2 inserts in chromosomal DNA of WT, Ku70−/−, Artemis−/− and LigIV−/− DT40 cells (26, 25, 24 and 27 insertions, respectively; Table S4). We previously showed that ∼40% of ZfL2-2 elements in the zebrafish genome had extra nucleotides at the 5′ junction, whereas ∼50% had microhomologies [29]. At the 3′ junction, on the other hand, ∼80% of these elements had microhomologies [29]. Similar tendencies were observed at both junctions of ZfL2-2 insertions in DT40 cells, and these tendencies were not altered by NHEJ defects (Table S5). Also, the length distribution of the 5′ and 3′ microhomologies did not differ between the WT and NHEJ-deficient DT40 cells (Figure S15). However, the ZfL2-2 insertions in Ku70−/− and Artemis−/− cells were significantly longer than those in WT cells (Figure 3A and 3B; P = 0.008 and 0.036, respectively). In particular, full-length elements were recovered only from NHEJ-deficient cells (Figure 3A, 3C). Indeed, the fraction of full-length insertions differed significantly between WT and Ku70−/− cells and between WT and Artemis−/− cells (P = 0.010 and 0.046, respectively). These results indicate that Ku70 and Artemis inhibit the generation of longer inserts in DT40 cells, suggesting that these NHEJ factors, at least in part, participate in LINE 5′ truncation.


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)

Characterization of ZfL2-2 insertions in DT40 cells.Abbreviations are as defined for Figure 1. (A) ZfL2-2 insertions isolated from DT40 cells. The top diagram shows the full-length ZfL2-2 element containing the mneoI400/ColE1 cassette (Cassette) in the 3′ UTR. ORF, open reading frame. Each horizontal line represents one of the 26, 25, 24 or 27 ZfL2-2 insertions isolated from the various DT40 cell lines. Blue lines represent insertions with a 5′ truncation. Red lines represent full-length insertions. The dashed line in Art−/− indicates a deletion. (B) A box-and-whisker plot shows the median (red line), the first and third quartiles, and the upper and lower limits of the length of insertions indicated in (A). P values less than 0.05 are indicated (Mann-Whitney U test). (C) Full-length vs. truncated elements. The ZfL2-2 insertions in (A) were categorized by the absence (Full) or presence (Truncated) of a 5′ truncation. The number of insertions identified is indicated inside each bar. P values less than 0.05 are indicated (two-sided Fisher's exact test). (D) Target site alterations. The ZfL2-2 insertions shown in (A) were categorized with regard to target site alterations. The number of insertions identified is indicated inside each bar. L-TST, long target site truncation (>20 bp). S-TST, short target site truncation (≤20 bp). BEJ, blunt end joining. S-TSD, short target site duplication (≤20 bp). L-TSD, long target site duplication (>20 bp). (E) Short target site alterations. The ZfL2-2 insertions with short target site alterations in (D) were compared. The number of insertions identified is indicated inside each bar. Abbreviations and definitions are as for panel D. P values less than 0.05 are indicated (Wilcoxon Rank Sum test).
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pgen-1000461-g003: Characterization of ZfL2-2 insertions in DT40 cells.Abbreviations are as defined for Figure 1. (A) ZfL2-2 insertions isolated from DT40 cells. The top diagram shows the full-length ZfL2-2 element containing the mneoI400/ColE1 cassette (Cassette) in the 3′ UTR. ORF, open reading frame. Each horizontal line represents one of the 26, 25, 24 or 27 ZfL2-2 insertions isolated from the various DT40 cell lines. Blue lines represent insertions with a 5′ truncation. Red lines represent full-length insertions. The dashed line in Art−/− indicates a deletion. (B) A box-and-whisker plot shows the median (red line), the first and third quartiles, and the upper and lower limits of the length of insertions indicated in (A). P values less than 0.05 are indicated (Mann-Whitney U test). (C) Full-length vs. truncated elements. The ZfL2-2 insertions in (A) were categorized by the absence (Full) or presence (Truncated) of a 5′ truncation. The number of insertions identified is indicated inside each bar. P values less than 0.05 are indicated (two-sided Fisher's exact test). (D) Target site alterations. The ZfL2-2 insertions shown in (A) were categorized with regard to target site alterations. The number of insertions identified is indicated inside each bar. L-TST, long target site truncation (>20 bp). S-TST, short target site truncation (≤20 bp). BEJ, blunt end joining. S-TSD, short target site duplication (≤20 bp). L-TSD, long target site duplication (>20 bp). (E) Short target site alterations. The ZfL2-2 insertions with short target site alterations in (D) were compared. The number of insertions identified is indicated inside each bar. Abbreviations and definitions are as for panel D. P values less than 0.05 are indicated (Wilcoxon Rank Sum test).
Mentions: To determine in which step of the retrotransposition reaction each NHEJ factor is involved, we determined and analyzed the 5′ and 3′ junction sequences of 102 ZfL2-2 inserts in chromosomal DNA of WT, Ku70−/−, Artemis−/− and LigIV−/− DT40 cells (26, 25, 24 and 27 insertions, respectively; Table S4). We previously showed that ∼40% of ZfL2-2 elements in the zebrafish genome had extra nucleotides at the 5′ junction, whereas ∼50% had microhomologies [29]. At the 3′ junction, on the other hand, ∼80% of these elements had microhomologies [29]. Similar tendencies were observed at both junctions of ZfL2-2 insertions in DT40 cells, and these tendencies were not altered by NHEJ defects (Table S5). Also, the length distribution of the 5′ and 3′ microhomologies did not differ between the WT and NHEJ-deficient DT40 cells (Figure S15). However, the ZfL2-2 insertions in Ku70−/− and Artemis−/− cells were significantly longer than those in WT cells (Figure 3A and 3B; P = 0.008 and 0.036, respectively). In particular, full-length elements were recovered only from NHEJ-deficient cells (Figure 3A, 3C). Indeed, the fraction of full-length insertions differed significantly between WT and Ku70−/− cells and between WT and Artemis−/− cells (P = 0.010 and 0.046, respectively). These results indicate that Ku70 and Artemis inhibit the generation of longer inserts in DT40 cells, suggesting that these NHEJ factors, at least in part, participate in LINE 5′ truncation.

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