Nucleic Acids Research Advance Access published online on April 19, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn178
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Structural Biology |
Crystal structures of I-SceI complexed to nicked DNA substrates: snapshots of intermediates along the DNA cleavage reaction pathway
1Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, 77030 and 2Department of Biochemistry, Purdue University, West Lafayette, IN, 47907, USA
*To whom correspondence should be addressed. Tel: +1 713 798 6564; Fax: +1 713 798 8516; Email: mmoure{at}bcm.tmc.edu
Received January 27, 2008. Revised March 26, 2008. Accepted March 28, 2008.
I-SceI is a homing endonuclease that specifically cleaves an 18-bp double-stranded DNA. I-SceI exhibits a strong preference for cleaving the bottom strand DNA. The published structure of I-SceI bound to an uncleaved DNA substrate provided a mechanism for bottom strand cleavage but not for top strand cleavage. To more fully elucidate the I-SceI catalytic mechanism, we determined the X-ray structures of I-SceI in complex with DNA substrates that are nicked in either the top or bottom strands. The structures resemble intermediates along the DNA cleavage reaction. In a structure containing a nick in the top strand, the spatial arrangement of metal ions is similar to that observed in the structure that contains uncleaved DNA, suggesting that cleavage of the bottom strand occurs by a common mechanism regardless of whether this strand is cleaved first or second. In the structure containing a nick in the bottom strand, a new metal binding site is present in the active site that cleaves the top strand. This new metal and a candidate nucleophilic water molecule are correctly positioned to cleave the top strand following bottom strand cleavage, providing a plausible mechanism for top strand cleavage.