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

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.