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Nucleic Acids Research Advance Access originally published online on August 20, 2008
Nucleic Acids Research 2008 36(16):5417-5425; doi:10.1093/nar/gkn528
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Nucleic Acids Research, 2008, Vol. 36, No. 16 5417-5425
© 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.

Nucleic Acid Enzymes

DNA base flipping by both members of the PspGI restriction–modification system

Michael A. Carpenter and Ashok S. Bhagwat*

Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, MI 48202, USA

*To whom correspondence should be addressed. Tel: +313 577 2547; Fax: +313 577 8822; Email: axb{at}chem.wayne.edu

Received May 1, 2008. Revised July 29, 2008. Accepted August 2, 2008.

The PspGI restriction–modification system recognizes the sequence CCWGG. R.PspGI cuts DNA before the first C in the cognate sequence and M.PspGI is thought to methylate N4 of one of the cytosines in the sequence. M.PspGI enhances fluorescence of 2-aminopurine in DNA if it replaces the second C in the sequence, while R.PspGI enhances fluorescence when the fluorophore replaces adenine in the central base pair. This strongly suggests that the methyltransferase flips the second C in the recognition sequence, while the endonuclease flips both bases in the central base pair out of the duplex. M.PspGI is the first N4-cytosine MTase for which biochemical evidence for base flipping has been presented. It is also the first type IIP methyltransferase whose catalytic activity is strongly stimulated by divalent metal ions. However, divalent metal ions are not required for its base-flipping activity. In contrast, these ions are required for both base flipping and catalysis by the endonuclease. The two enzymes have similar temperature profiles for base flipping and optimal flipping occurs at temperatures substantially below the growth temperature of the source organism for PspGI and for the catalytic activity of endonuclease. We discuss the implications of these results for DNA binding by these enzymes and their evolutionary origin.


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