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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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Identification of sequences involved in the cleavage of the target sites. Nicking activities of the target sites on mutated substrates were examined (A, bottom strand cleavage; B, top strand cleavage). Mutated substrates contained TT or AA substitution (boxed) and a mutated position of the substrates was sequentially changed. Each substrate was numbered and CTRL represents a non-mutated control substrate. Nicked strands of mutated substrates are shown at the lower left of each panel and mutated bases were boxed. The solid and open arrowheads indicate the cleavage sites on the top and bottom strands, respectively. Three picomoles (for bottom strand cleavage) or 1 pmol (for top strand cleavage) of the mutated substrate was incubated with R1EN and electrophoresed. Gel bands around the cleavage products were shown at the top of each panel. The size marker has the same sequence as the expected cleaved product. The cleavage products nicked at the target site were quantified and the percentage of the cleavage product relative to that of a control substrate was shown at the lower bottom of each panel. The results of three independent experiments were averaged and error bars show S.E.
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Figure 2: Identification of sequences involved in the cleavage of the target sites. Nicking activities of the target sites on mutated substrates were examined (A, bottom strand cleavage; B, top strand cleavage). Mutated substrates contained TT or AA substitution (boxed) and a mutated position of the substrates was sequentially changed. Each substrate was numbered and CTRL represents a non-mutated control substrate. Nicked strands of mutated substrates are shown at the lower left of each panel and mutated bases were boxed. The solid and open arrowheads indicate the cleavage sites on the top and bottom strands, respectively. Three picomoles (for bottom strand cleavage) or 1 pmol (for top strand cleavage) of the mutated substrate was incubated with R1EN and electrophoresed. Gel bands around the cleavage products were shown at the top of each panel. The size marker has the same sequence as the expected cleaved product. The cleavage products nicked at the target site were quantified and the percentage of the cleavage product relative to that of a control substrate was shown at the lower bottom of each panel. The results of three independent experiments were averaged and error bars show S.E.

Mentions: We next determined the sequences required for the sequence-specific nicks on the bottom or top strand. A series of mutated substrates was prepared. Each substrate contained a 2-bp substitution, TT or AA, on the bottom or top strand, respectively. The mutated site was gradually moved in 2-bp steps from 5′ to 3′ around the cleavage site. The nicking activity on the mutated substrate was tested and compared with that on the non-mutated substrate. During bottom-strand cleavage, no significant change in nicking activity was detected on six substrates (lanes B1 to B3 and B9 to B11, Figure 2A). However, mutations within 5 bp of the cleavage site, either upstream or downstream, resulted in a severe reduction in nicking activity (lanes B4 to B8, Figure 2A), suggesting that this 10-bp region is important for the sequence-specific cleavage on the bottom strand. On the substrates of lanes B5 and B8, we observed strong signals at positions 4-bp shorter (B5#) and 2-bp longer (B8*) than the original cleavage site, respectively. This indicates that these mutations result in novel cleavage sites, other than the original site targeted by R1Bm EN.Figure 2.


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)

Identification of sequences involved in the cleavage of the target sites. Nicking activities of the target sites on mutated substrates were examined (A, bottom strand cleavage; B, top strand cleavage). Mutated substrates contained TT or AA substitution (boxed) and a mutated position of the substrates was sequentially changed. Each substrate was numbered and CTRL represents a non-mutated control substrate. Nicked strands of mutated substrates are shown at the lower left of each panel and mutated bases were boxed. The solid and open arrowheads indicate the cleavage sites on the top and bottom strands, respectively. Three picomoles (for bottom strand cleavage) or 1 pmol (for top strand cleavage) of the mutated substrate was incubated with R1EN and electrophoresed. Gel bands around the cleavage products were shown at the top of each panel. The size marker has the same sequence as the expected cleaved product. The cleavage products nicked at the target site were quantified and the percentage of the cleavage product relative to that of a control substrate was shown at the lower bottom of each panel. The results of three independent experiments were averaged and error bars show S.E.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Identification of sequences involved in the cleavage of the target sites. Nicking activities of the target sites on mutated substrates were examined (A, bottom strand cleavage; B, top strand cleavage). Mutated substrates contained TT or AA substitution (boxed) and a mutated position of the substrates was sequentially changed. Each substrate was numbered and CTRL represents a non-mutated control substrate. Nicked strands of mutated substrates are shown at the lower left of each panel and mutated bases were boxed. The solid and open arrowheads indicate the cleavage sites on the top and bottom strands, respectively. Three picomoles (for bottom strand cleavage) or 1 pmol (for top strand cleavage) of the mutated substrate was incubated with R1EN and electrophoresed. Gel bands around the cleavage products were shown at the top of each panel. The size marker has the same sequence as the expected cleaved product. The cleavage products nicked at the target site were quantified and the percentage of the cleavage product relative to that of a control substrate was shown at the lower bottom of each panel. The results of three independent experiments were averaged and error bars show S.E.
Mentions: We next determined the sequences required for the sequence-specific nicks on the bottom or top strand. A series of mutated substrates was prepared. Each substrate contained a 2-bp substitution, TT or AA, on the bottom or top strand, respectively. The mutated site was gradually moved in 2-bp steps from 5′ to 3′ around the cleavage site. The nicking activity on the mutated substrate was tested and compared with that on the non-mutated substrate. During bottom-strand cleavage, no significant change in nicking activity was detected on six substrates (lanes B1 to B3 and B9 to B11, Figure 2A). However, mutations within 5 bp of the cleavage site, either upstream or downstream, resulted in a severe reduction in nicking activity (lanes B4 to B8, Figure 2A), suggesting that this 10-bp region is important for the sequence-specific cleavage on the bottom strand. On the substrates of lanes B5 and B8, we observed strong signals at positions 4-bp shorter (B5#) and 2-bp longer (B8*) than the original cleavage site, respectively. This indicates that these mutations result in novel cleavage sites, other than the original site targeted by R1Bm EN.Figure 2.

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