Nucleic Acids Research Advance Access originally published online on May 30, 2007
Nucleic Acids Research 2007 35(12):3918-3927; doi:10.1093/nar/gkm397
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Nucleic Acids Research, 2007, Vol. 35, No. 12 3918-3927
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Molecular Biology |
Characterization of the sequence specificity of the R1Bm endonuclease domain by structural and biochemical studies
1Graduate School of Systems Life Sciences, Kyushu University, Fukuoka 812-8582, Japan, 2Graduate School of Integrated Science, Yokohama City University, Yokohama 230-0045, Japan, 3Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Bioscience Building 501, Kashiwa, Chiba 277-8562, Japan, 4Graduate School of Engineering Kyoto University, Kyoto 615-8510, Japan and 5CREST, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan
*To whom correspondence should be addressed. Tel: +81-4-7136-3659; Fax: +81-4-7136-3660; Email: haruh{at}k.u-tokyo.ac.jp
Received December 1, 2006. Revised April 25, 2007. Accepted May 1, 2007.
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 Å resolution. Its structure was basically similar to AP endonuclease family, but had a special ß-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.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.