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

Nucleic Acids Research
Michael A Carpenter, Ashok S Bhagwat

Abstract

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 i...Continue Reading

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Citations

Oct 3, 2008·Nucleic Acids Research·Roman H SzczepanowskiMatthias Bochtler
Sep 11, 2009·Nucleic Acids Research·Robert K NeelyAnita C Jones
May 8, 2014·The Journal of Physical Chemistry Letters·Elvin A AlemánDavid Rueda

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Methods Mentioned

BETA
deamination
Protein Assay
Fluorescence

Software Mentioned

BLAST

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