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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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Mutagenesis study of R1Bm EN. (A) Positions of residues mutated in this study. The positions of residues substituted with alanine are indicated as line models (magenta). His-209 is also depicted to indicate the catalytic site. (B) Bottom strand cleavage by R1Bm EN mutants. Three picomoles of the substrate was cleaved with wild-type R1Bm EN and the mutants. The solid arrowheads indicate the target site cleavage of the bottom strand. (C) Top strand cleavage by R1Bm EN mutants. One picomole of the substrate was used. The open arrowheads indicate the target site cleavage of the top strand. (D) Nicking activities of R1Bm EN and the mutants. The cleavage products nicked at the target site were quantified and the percentage of the cleavage product relative to that of wild-type R1Bm EN was shown in each strand cleavage. The mutants representing <20% of wild-type activity in both strand cleavage are underlined in magenta and the mutants showing the great reduction of the nicking activity only in bottom strand cleavage are underlined in blue. The results of three independent experiments were averaged and error bars show S.E. (E) Cleavage of the bottom strand by R1Bm EN mutants under the vigorous condition. The cleaved bands corresponding to Figure 1B are indicated. Asterisk indicates the abnormal band observed in Y98A mutant.
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Figure 6: Mutagenesis study of R1Bm EN. (A) Positions of residues mutated in this study. The positions of residues substituted with alanine are indicated as line models (magenta). His-209 is also depicted to indicate the catalytic site. (B) Bottom strand cleavage by R1Bm EN mutants. Three picomoles of the substrate was cleaved with wild-type R1Bm EN and the mutants. The solid arrowheads indicate the target site cleavage of the bottom strand. (C) Top strand cleavage by R1Bm EN mutants. One picomole of the substrate was used. The open arrowheads indicate the target site cleavage of the top strand. (D) Nicking activities of R1Bm EN and the mutants. The cleavage products nicked at the target site were quantified and the percentage of the cleavage product relative to that of wild-type R1Bm EN was shown in each strand cleavage. The mutants representing <20% of wild-type activity in both strand cleavage are underlined in magenta and the mutants showing the great reduction of the nicking activity only in bottom strand cleavage are underlined in blue. The results of three independent experiments were averaged and error bars show S.E. (E) Cleavage of the bottom strand by R1Bm EN mutants under the vigorous condition. The cleaved bands corresponding to Figure 1B are indicated. Asterisk indicates the abnormal band observed in Y98A mutant.

Mentions: The 32P-labeled substrates containing the R1Bm target site were prepared exactly as previously described (18). The 40-bp top or bottom strand oligonucleotides were radio-labeled, annealed with the complementary non-labeled oligonucleotides and gel-purified. The cold substrates were also prepared by annealing of non-labeled top and bottom strand oligonucleotides. The mixture of labeled and non-labeled substrates was used for the cleavage reaction. The reaction mixture containing 50 mM PIPES-NaOH at pH 6.0, 17.5 mM NaCl, 1 mM MgCl2, 200 ng of purified proteins and 1 or 3 pmol of substrate DNA (the molar ratio of protein:DNA is 15:2 or 15:6) in a total volume of 10 μl was incubated at 25°C for 60 min. The reaction was stopped by the addition of 10 μl of denaturing solution (95% formamide, 50 mM EDTA, 0.01% bromophenol blue). The reaction product was denatured for 3 min at 95°C, immediately chilled on ice, and separated on a 30% polyacrylamide denaturing gel. The cleavage efficiency was quantified with BAS 5000 imaging analyzer system (Fujifilm). In the experiment of Figure 6E, oligonucleotide cleavage assays were performed with the reaction mixture containing 10 mM MgCl2 and 0.1 pmol of substrate DNA, and incubation time was increased from 1 to 5 h.


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)

Mutagenesis study of R1Bm EN. (A) Positions of residues mutated in this study. The positions of residues substituted with alanine are indicated as line models (magenta). His-209 is also depicted to indicate the catalytic site. (B) Bottom strand cleavage by R1Bm EN mutants. Three picomoles of the substrate was cleaved with wild-type R1Bm EN and the mutants. The solid arrowheads indicate the target site cleavage of the bottom strand. (C) Top strand cleavage by R1Bm EN mutants. One picomole of the substrate was used. The open arrowheads indicate the target site cleavage of the top strand. (D) Nicking activities of R1Bm EN and the mutants. The cleavage products nicked at the target site were quantified and the percentage of the cleavage product relative to that of wild-type R1Bm EN was shown in each strand cleavage. The mutants representing <20% of wild-type activity in both strand cleavage are underlined in magenta and the mutants showing the great reduction of the nicking activity only in bottom strand cleavage are underlined in blue. The results of three independent experiments were averaged and error bars show S.E. (E) Cleavage of the bottom strand by R1Bm EN mutants under the vigorous condition. The cleaved bands corresponding to Figure 1B are indicated. Asterisk indicates the abnormal band observed in Y98A mutant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Mutagenesis study of R1Bm EN. (A) Positions of residues mutated in this study. The positions of residues substituted with alanine are indicated as line models (magenta). His-209 is also depicted to indicate the catalytic site. (B) Bottom strand cleavage by R1Bm EN mutants. Three picomoles of the substrate was cleaved with wild-type R1Bm EN and the mutants. The solid arrowheads indicate the target site cleavage of the bottom strand. (C) Top strand cleavage by R1Bm EN mutants. One picomole of the substrate was used. The open arrowheads indicate the target site cleavage of the top strand. (D) Nicking activities of R1Bm EN and the mutants. The cleavage products nicked at the target site were quantified and the percentage of the cleavage product relative to that of wild-type R1Bm EN was shown in each strand cleavage. The mutants representing <20% of wild-type activity in both strand cleavage are underlined in magenta and the mutants showing the great reduction of the nicking activity only in bottom strand cleavage are underlined in blue. The results of three independent experiments were averaged and error bars show S.E. (E) Cleavage of the bottom strand by R1Bm EN mutants under the vigorous condition. The cleaved bands corresponding to Figure 1B are indicated. Asterisk indicates the abnormal band observed in Y98A mutant.
Mentions: The 32P-labeled substrates containing the R1Bm target site were prepared exactly as previously described (18). The 40-bp top or bottom strand oligonucleotides were radio-labeled, annealed with the complementary non-labeled oligonucleotides and gel-purified. The cold substrates were also prepared by annealing of non-labeled top and bottom strand oligonucleotides. The mixture of labeled and non-labeled substrates was used for the cleavage reaction. The reaction mixture containing 50 mM PIPES-NaOH at pH 6.0, 17.5 mM NaCl, 1 mM MgCl2, 200 ng of purified proteins and 1 or 3 pmol of substrate DNA (the molar ratio of protein:DNA is 15:2 or 15:6) in a total volume of 10 μl was incubated at 25°C for 60 min. The reaction was stopped by the addition of 10 μl of denaturing solution (95% formamide, 50 mM EDTA, 0.01% bromophenol blue). The reaction product was denatured for 3 min at 95°C, immediately chilled on ice, and separated on a 30% polyacrylamide denaturing gel. The cleavage efficiency was quantified with BAS 5000 imaging analyzer system (Fujifilm). In the experiment of Figure 6E, oligonucleotide cleavage assays were performed with the reaction mixture containing 10 mM MgCl2 and 0.1 pmol of substrate DNA, and incubation time was increased from 1 to 5 h.

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