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Related Experiment Videos

Substrate recognition by Escherichia coli MutY using substrate analogs.

C L Chepanoske1, S L Porello, T Fujiwara

  • 1Department of Chemistry, University of Utah, Salt Lake City, UT 84112, USA.

Nucleic Acids Research
|August 24, 1999
PubMed
Summary

The Escherichia coli MutY enzyme recognizes DNA mismatches using stable substrate analogs. This reveals a nucleotide flipping mechanism for DNA repair, aiding in understanding MutY

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Area of Science:

  • Molecular Biology
  • DNA Repair Mechanisms
  • Enzymology

Background:

  • Escherichia coli adenine glycosylase MutY repairs 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG):A and G:A mispairs in DNA.
  • Understanding MutY's DNA damage recognition requires stable MutY-DNA complexes.
  • Substrate analogs resistant to glycosylase activity are used to study these interactions.

Purpose of the Study:

  • To investigate the interaction of MutY with DNA duplexes containing 2'-deoxyadenosine analogs: 2'-deoxy-2'-fluoroadenosine (FA), 2'-deoxyaristeromycin (R), and 2'-deoxyformycin A (F).
  • To characterize the binding and conformational changes induced by MutY upon interaction with these analogs.

Main Methods:

  • Biochemical characterization of MutY-DNA complexes using substrate analogs.
  • MPE-Fe(II) hydroxyl radical footprinting to map MutY binding sites.

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  • DMS footprinting to assess DNA conformational changes upon MutY binding.

    • Main Results:

    • MutY binds with high affinity to DNA duplexes containing FA, R, or F analogs opposite G and OG, but shows no enzymatic processing.
    • Hydroxyl radical footprinting protected a nine base pair region around the OG:FA mispair.
    • DMS footprinting indicated a conformational change, exposing the analog-containing strand and suggesting a nucleotide flipping mechanism.

    Conclusions:

    • Duplex substrates containing FA, R, or F analogs serve as excellent mimics for studying MutY recognition of damaged and mismatched base pairs.
    • The results support a nucleotide flipping mechanism for DNA damage recognition by MutY.
    • These stable analogs facilitate structural and biochemical studies of DNA repair enzymes.