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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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Structural comparison of R1Bm EN. (A) Stereoview representation of superposed backbone model of R1Bm EN (green), TRAS1-EN (PDB entry:1WDU, magenta) and L1-EN (PDB entry:1VYB, cyan). β-hairpin region (β8–β9) and α3 are indicated. (B) Close up view of superposed active site of R1Bm EN (green), TRAS1-EN (magenta), L1-EN (cyan) and human APE1 (PDB entry: 1DEW, pale orange). Residue names and numbers of R1Bm EN are shown.
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Figure 5: Structural comparison of R1Bm EN. (A) Stereoview representation of superposed backbone model of R1Bm EN (green), TRAS1-EN (PDB entry:1WDU, magenta) and L1-EN (PDB entry:1VYB, cyan). β-hairpin region (β8–β9) and α3 are indicated. (B) Close up view of superposed active site of R1Bm EN (green), TRAS1-EN (magenta), L1-EN (cyan) and human APE1 (PDB entry: 1DEW, pale orange). Residue names and numbers of R1Bm EN are shown.

Mentions: The overall structure of R1Bm EN is a four-layered α/β sandwiched fold (Figure 4A), and is almost identical to that of other known EN domains of the APE-type LINEs, TRAS1 EN and L1 EN. The root-mean-square differences of the Cα atoms of the core β-sheets (71 residues) between R1Bm EN versus TRAS1 EN and L1 EN are 0.68 Å and 2.21 Å, respectively. Furthermore, three α-helices located outside the β-sheets also fit well between R1Bm EN and TRAS1 EN (Figure 5A), indicating that the R1Bm EN structure resembles the TRAS1 EN structure more than the L1 EN structure. This observation is consistent with previous phylogenetic analyses of the LINE family, which demonstrated that TRAS1 and R1Bm are in the same clade, whereas L1 is not (20).Figure 5.


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)

Structural comparison of R1Bm EN. (A) Stereoview representation of superposed backbone model of R1Bm EN (green), TRAS1-EN (PDB entry:1WDU, magenta) and L1-EN (PDB entry:1VYB, cyan). β-hairpin region (β8–β9) and α3 are indicated. (B) Close up view of superposed active site of R1Bm EN (green), TRAS1-EN (magenta), L1-EN (cyan) and human APE1 (PDB entry: 1DEW, pale orange). Residue names and numbers of R1Bm EN are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Structural comparison of R1Bm EN. (A) Stereoview representation of superposed backbone model of R1Bm EN (green), TRAS1-EN (PDB entry:1WDU, magenta) and L1-EN (PDB entry:1VYB, cyan). β-hairpin region (β8–β9) and α3 are indicated. (B) Close up view of superposed active site of R1Bm EN (green), TRAS1-EN (magenta), L1-EN (cyan) and human APE1 (PDB entry: 1DEW, pale orange). Residue names and numbers of R1Bm EN are shown.
Mentions: The overall structure of R1Bm EN is a four-layered α/β sandwiched fold (Figure 4A), and is almost identical to that of other known EN domains of the APE-type LINEs, TRAS1 EN and L1 EN. The root-mean-square differences of the Cα atoms of the core β-sheets (71 residues) between R1Bm EN versus TRAS1 EN and L1 EN are 0.68 Å and 2.21 Å, respectively. Furthermore, three α-helices located outside the β-sheets also fit well between R1Bm EN and TRAS1 EN (Figure 5A), indicating that the R1Bm EN structure resembles the TRAS1 EN structure more than the L1 EN structure. This observation is consistent with previous phylogenetic analyses of the LINE family, which demonstrated that TRAS1 and R1Bm are in the same clade, whereas L1 is not (20).Figure 5.

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