Nucleic Acids Research Advance Access originally published online on August 13, 2008
Nucleic Acids Research 2008 36(16):5405-5416; doi:10.1093/nar/gkn510
Nucleic Acids Research, 2008, Vol. 36, No. 16 5405-5416
© 2008 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.
Structural Biology |
The structure of SgrAI bound to DNA; recognition of an 8 base pair target
1Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CA 94025, 2Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, AZ 85721 and 3New England Biolabs, 240 County Road Ipswich, MA 01938-2723, USA
*To whom correspondence should be addressed. Tel: +520 626 3828; Fax: +520 621 9288; Email: nhorton{at}u.arizona.edu
Received July 3, 2008. Revised July 19, 2008. Accepted July 24, 2008.
The three-dimensional X-ray crystal structure of the ‘rare cutting’ type II restriction endonuclease SgrAI bound to cognate DNA is presented. SgrAI forms a dimer bound to one duplex of DNA. Two Ca2+ bind in the enzyme active site, with one ion at the interface between the protein and DNA, and the second bound distal from the DNA. These sites are differentially occupied by Mn2+, with strong binding at the protein–DNA interface, but only partial occupancy of the distal site. The DNA remains uncleaved in the structures from crystals grown in the presence of either divalent cation. The structure of the dimer of SgrAI is similar to those of Cfr10I, Bse634I and NgoMIV, however no tetrameric structure of SgrAI is observed. DNA contacts to the central CCGG base pairs of the SgrAI canonical target sequence (CR|CCGGYG, | marks the site of cleavage) are found to be very similar to those in the NgoMIV/DNA structure (target sequence G|CCGGC). Specificity at the degenerate YR base pairs of the SgrAI sequence may occur via indirect readout using DNA distortion. Recognition of the outer GC base pairs occurs through a single contact to the G from an arginine side chain located in a region unique to SgrAI.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
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