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Characterization of the sequence specificity of the R1Bm endonuclease domain by structural and biochemical studies.

Maita N, Aoyagi H, Osanai M, Shirakawa M, Fujiwara H - Nucleic Acids Res. (2007)

Bottom Line: Point-mutations on the DNA binding surface of R1Bm EN significantly decreased the cleavage activities, but did not affect the sequence recognition in most residues.However, two mutants Y98A and N180A had altered cleavage patterns, suggesting an important role of these residues (Y98 and N180) for the sequence recognition of R1Bm EN.In addition, Y98A mutant showed another cleavage pattern, that implies de novo design of novel sequence-specific EN.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Systems Life Sciences, Kyushu University, Fukuoka 812-8582, Japan.

ABSTRACT
R1Bm is a long interspersed element (LINE) inserted into a specific sequence within 28S rDNA of the silkworm genome. Of two open reading frames (ORFs) of R1Bm, ORF2 encodes a reverse transcriptase (RT) and an endonuclease (EN) domain which digests specifically both top and bottom strand of the target sequence in 28S rDNA. To elucidate the sequence specificity of EN domain of R1Bm (R1Bm EN), we examined the cleavage tendency for the target sequences, and found that 5'-A(G/C)(A/T)!(A/G)T-3' is the consensus sequence (! = cleavage site). We also determined the crystal structure of R1Bm EN at 2.0 A resolution. Its structure was basically similar to AP endonuclease family, but had a special beta-hairpin at the edge of the DNA binding surface, which is a common feature among EN of LINEs. Point-mutations on the DNA binding surface of R1Bm EN significantly decreased the cleavage activities, but did not affect the sequence recognition in most residues. However, two mutants Y98A and N180A had altered cleavage patterns, suggesting an important role of these residues (Y98 and N180) for the sequence recognition of R1Bm EN. In addition, Y98A mutant showed another cleavage pattern, that implies de novo design of novel sequence-specific EN.

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Crystal structure of R1Bm EN. (A) Cartoon representation of R1Bm EN (5–219). The characteristic β-hairpin region (β8–β9) and α3 are indicated. (B) The sigma-weighted 2Fo-Fc electron-density map showing around the active site of R1Bm EN. The map was contoured at 1.5σ. (C) The sequence-alignment of R1Bm EN, TRAS1-EN, L1-EN (human) and human APE1. The secondary structure of R1Bm EN is indicated above the amino acid sequence. Residues conserved among all or most APE family are indicated as red or black bold cases, respectively. Residues substituted to alanine in mutagenesis study are highlighted by green. All figures were prepared by PyMOL (DeLano Scientific, http://pymol.sourceforge.net/).
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Figure 4: Crystal structure of R1Bm EN. (A) Cartoon representation of R1Bm EN (5–219). The characteristic β-hairpin region (β8–β9) and α3 are indicated. (B) The sigma-weighted 2Fo-Fc electron-density map showing around the active site of R1Bm EN. The map was contoured at 1.5σ. (C) The sequence-alignment of R1Bm EN, TRAS1-EN, L1-EN (human) and human APE1. The secondary structure of R1Bm EN is indicated above the amino acid sequence. Residues conserved among all or most APE family are indicated as red or black bold cases, respectively. Residues substituted to alanine in mutagenesis study are highlighted by green. All figures were prepared by PyMOL (DeLano Scientific, http://pymol.sourceforge.net/).

Mentions: To clarify its sequence specificity at the atomic level, R1Bm EN (residues 1–220) were expressed, purified and crystallized (‘Materials and Methods’). The crystal structure of R1Bm EN, the final model for which contained 215 amino acids (residues 5–219), was resolved by molecular replacement at 2.0 Å resolution, with values for R-factor and R-free of 19.5% and 21.1%, respectively (Figures 4A and B). The asymmetric component of the crystal is a monomer. Furthermore, in gel filtration chromatography during the purification, R1Bm EN eluted with a fraction corresponding to 22 kDa, therefore, R1Bm EN does not multimerize under this condition (Figure S3).Figure 4.


Characterization of the sequence specificity of the R1Bm endonuclease domain by structural and biochemical studies.

Maita N, Aoyagi H, Osanai M, Shirakawa M, Fujiwara H - Nucleic Acids Res. (2007)

Crystal structure of R1Bm EN. (A) Cartoon representation of R1Bm EN (5–219). The characteristic β-hairpin region (β8–β9) and α3 are indicated. (B) The sigma-weighted 2Fo-Fc electron-density map showing around the active site of R1Bm EN. The map was contoured at 1.5σ. (C) The sequence-alignment of R1Bm EN, TRAS1-EN, L1-EN (human) and human APE1. The secondary structure of R1Bm EN is indicated above the amino acid sequence. Residues conserved among all or most APE family are indicated as red or black bold cases, respectively. Residues substituted to alanine in mutagenesis study are highlighted by green. All figures were prepared by PyMOL (DeLano Scientific, http://pymol.sourceforge.net/).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919474&req=5

Figure 4: Crystal structure of R1Bm EN. (A) Cartoon representation of R1Bm EN (5–219). The characteristic β-hairpin region (β8–β9) and α3 are indicated. (B) The sigma-weighted 2Fo-Fc electron-density map showing around the active site of R1Bm EN. The map was contoured at 1.5σ. (C) The sequence-alignment of R1Bm EN, TRAS1-EN, L1-EN (human) and human APE1. The secondary structure of R1Bm EN is indicated above the amino acid sequence. Residues conserved among all or most APE family are indicated as red or black bold cases, respectively. Residues substituted to alanine in mutagenesis study are highlighted by green. All figures were prepared by PyMOL (DeLano Scientific, http://pymol.sourceforge.net/).
Mentions: To clarify its sequence specificity at the atomic level, R1Bm EN (residues 1–220) were expressed, purified and crystallized (‘Materials and Methods’). The crystal structure of R1Bm EN, the final model for which contained 215 amino acids (residues 5–219), was resolved by molecular replacement at 2.0 Å resolution, with values for R-factor and R-free of 19.5% and 21.1%, respectively (Figures 4A and B). The asymmetric component of the crystal is a monomer. Furthermore, in gel filtration chromatography during the purification, R1Bm EN eluted with a fraction corresponding to 22 kDa, therefore, R1Bm EN does not multimerize under this condition (Figure S3).Figure 4.

Bottom Line: Point-mutations on the DNA binding surface of R1Bm EN significantly decreased the cleavage activities, but did not affect the sequence recognition in most residues.However, two mutants Y98A and N180A had altered cleavage patterns, suggesting an important role of these residues (Y98 and N180) for the sequence recognition of R1Bm EN.In addition, Y98A mutant showed another cleavage pattern, that implies de novo design of novel sequence-specific EN.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Systems Life Sciences, Kyushu University, Fukuoka 812-8582, Japan.

ABSTRACT
R1Bm is a long interspersed element (LINE) inserted into a specific sequence within 28S rDNA of the silkworm genome. Of two open reading frames (ORFs) of R1Bm, ORF2 encodes a reverse transcriptase (RT) and an endonuclease (EN) domain which digests specifically both top and bottom strand of the target sequence in 28S rDNA. To elucidate the sequence specificity of EN domain of R1Bm (R1Bm EN), we examined the cleavage tendency for the target sequences, and found that 5'-A(G/C)(A/T)!(A/G)T-3' is the consensus sequence (! = cleavage site). We also determined the crystal structure of R1Bm EN at 2.0 A resolution. Its structure was basically similar to AP endonuclease family, but had a special beta-hairpin at the edge of the DNA binding surface, which is a common feature among EN of LINEs. Point-mutations on the DNA binding surface of R1Bm EN significantly decreased the cleavage activities, but did not affect the sequence recognition in most residues. However, two mutants Y98A and N180A had altered cleavage patterns, suggesting an important role of these residues (Y98 and N180) for the sequence recognition of R1Bm EN. In addition, Y98A mutant showed another cleavage pattern, that implies de novo design of novel sequence-specific EN.

Show MeSH
Related in: MedlinePlus