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DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling
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Central base pair flipping and discrimination by PspGI.

Roman H Szczepanowski1, Michael A Carpenter, Honorata Czapinska

  • 1International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland.

Nucleic Acids Research
|October 3, 2008
PubMed
Summary

PspGI restriction enzyme prefers A/T over G/C at its target DNA site by flipping bases into enzyme pockets. This base flipping mechanism contributes to DNA sequence discrimination.

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Area of Science:

  • Molecular Biology
  • Structural Biology
  • Enzymology

Background:

  • Restriction endonucleases are enzymes that cleave DNA at specific recognition sites.
  • PspGI is a restriction enzyme recognizing a CCNGG pentameric sequence.
  • PspGI exhibits specificity for the central base pair, preferring A/T over G/C, unlike Ecl18kI.

Purpose of the Study:

  • To elucidate the structural basis of PspGI's sequence specificity.
  • To understand the mechanism of base discrimination in DNA recognition by restriction enzymes.

Main Methods:

  • X-ray crystallography
  • Determination of the crystal structure of PspGI bound to target DNA at 1.7 Å resolution.

Main Results:

  • The central bases of the recognition sequence are extruded and flipped into PspGI pockets.
  • Flipped adenine adopts an unfavorable syn conformation, while flipped thymine remains in the anti conformation.
  • PspGI pockets are selected against guanine and cytosine, contributing to A/T preference.

Conclusions:

  • Base flipping contributes to discrimination between normal DNA bases.
  • This study reveals a novel role for base flipping in sequence recognition beyond modification and repair.
  • Structural insights into PspGI provide a mechanism for understanding enzyme-DNA interactions and specificity.