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DNA determinants in sequence-specific recognition by XmaI endonuclease

B E Withers1, J C Dunbar

  • 1Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI 48201, USA.

Nucleic Acids Research
|September 11, 1995
PubMed
Summary

The XmaI endonuclease binds DNA specifically, even without magnesium, interacting with key DNA bases and phosphates. This enzyme shares DNA recognition determinants with its neoschizomer, SmaI.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Enzymology

Background:

  • XmaI is a restriction endonuclease that cleaves the specific DNA sequence 5'-CCCGGG-3'.
  • Magnesium ions are essential for the catalytic activity of many endonucleases, but their role in DNA binding is not always fully understood.

Purpose of the Study:

  • To investigate the DNA binding properties of XmaI endonuclease in the absence of magnesium.
  • To elucidate the specific DNA-protein interactions involved in XmaI recognition.
  • To compare the DNA binding determinants of XmaI with its neoschizomer, SmaI.

Main Methods:

  • DNA-binding assays to determine the association constant.
  • Competition assays to assess binding specificity.
  • Missing nucleoside analysis and dimethylsulfate interference footprinting to identify interacting DNA residues.

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Main Results:

  • XmaI forms stable, specific DNA-protein complexes in the absence of magnesium, with a high affinity for its cognate site (K(a) = 1.2 x 10(9)/M).
  • The enzyme interacts with cytosine and guanine bases within the CCCGGG recognition sequence.
  • Major interactions occur with phosphates 5' to the guanines in the DNA backbone.
  • XmaI and SmaI share common DNA recognition determinants.

Conclusions:

  • XmaI exhibits high-affinity, sequence-specific DNA binding independent of magnesium.
  • The enzyme recognizes specific bases and backbone phosphates within its target sequence.
  • XmaI and SmaI utilize similar DNA interaction mechanisms despite potential differences in cleavage sites.