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Updated: Feb 14, 2026

Strand-Specific Analysis of Proteins at Replicating DNA Strands by Enrichment and Sequencing of Protein-Associated Nascent DNA Method
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Strand-Specific Analysis of Proteins at Replicating DNA Strands by Enrichment and Sequencing of Protein-Associated Nascent DNA Method

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Selection of novel, specific single-stranded DNA sequences by Flp, a duplex-specific DNA binding protein

X D Zhu1, P D Sadowski

  • 1Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada.

Nucleic Acids Research
|April 4, 1998
PubMed

Insights

The Flp recombinase, typically binding double-stranded DNA, unexpectedly binds single-stranded DNA with high affinity. This novel binding activity, also observed in Cre recombinase, expands our understanding of integrase family proteins.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Flp recombinase is a well-characterized site-specific recombinase belonging to the integrase family.
  • Flp is known to bind double-stranded DNA (dsDNA) specifically to the FRT site.
  • Integrase family recombinases are crucial tools in genetic engineering and molecular biology.

Purpose of the Study:

  • To investigate potential novel DNA binding activities of Flp recombinase beyond its known dsDNA binding.
  • To characterize the binding of Flp to single-stranded oligonucleotides (ssDNA).
  • To determine if this ssDNA binding activity is conserved in other integrase family members like Cre.

Main Methods:

  • In vitro binding site selection using a random pool of oligonucleotides.
  • Characterization of Flp binding affinity to ssDNA and dsDNA competitors.
  • Analysis of the functional domain responsible for ssDNA binding.
  • Comparative analysis of ssDNA binding in Cre recombinase.

Main Results:

  • Flp recombinase unexpectedly binds to specific single-stranded oligonucleotides with high affinity.
  • The minimal active ssDNA length is 29 nucleotides.
  • The ssDNA binding activity resides in the same C-terminal 32 kDa domain as dsDNA binding.
  • Cre recombinase also demonstrates similar ssDNA binding capabilities.

Conclusions:

  • Flp recombinase exhibits a previously unrecognized high-affinity binding to single-stranded DNA.
  • This ssDNA binding is sequence-specific and distinct from its FRT site recognition.
  • The conserved ssDNA binding in Cre suggests a broader role for this activity within the integrase family.

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