Crystal structures of I-SceI complexed to nicked DNA substrates: snapshots of intermediates along the DNA cleavage reaction pathway.

Nucleic Acids Research
Carmen M MoureFlorante A Quiocho

Abstract

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.

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Citations

Sep 22, 2010·Quarterly Reviews of Biophysics·Wei Yang
Nov 1, 2008·Proceedings of the National Academy of Sciences of the United States of America·María José MarcaidaGuillermo Montoya
Oct 26, 2011·Future Medicinal Chemistry·Béla Gyurcsik, Anikó Czene
Mar 12, 2009·Proceedings of the National Academy of Sciences of the United States of America·Audrey McConnell SmithBarry L Stoddard
Nov 17, 2009·Cellular and Molecular Life Sciences : CMLS·Maria J MarcaidaGuillermo Montoya
Aug 25, 2009·Current Opinion in Biotechnology·Frank Buchholz
Oct 9, 2009·The Plant Journal : for Cell and Molecular Biology·Huirong GaoL Alexander Lyznik
Dec 3, 2014·Biotechnology Advances·Tong SiHuimin Zhao
Oct 30, 2010·Journal of Molecular Biology·Rakesh JoshiFrederick S Gimble
Dec 9, 2014·Nature Structural & Molecular Biology·Rafael MolinaGuillermo Montoya
Oct 5, 2016·Nucleic Acids Research·Giulia Bastianello, Hiroshi Arakawa

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Methods Mentioned

BETA
X-ray

Software Mentioned

TREK
HKL200
CNS
MOLREP

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