The Flp recombinase encoded
by the 2 µm plasmid of yeast is a key component of the plasmid copy
number control mechanism. The recombination reaction is completed in two
steps, and requires the action of four Flp monomers. Each step involves
the phosphorylation and dephosphorylation, via nucleotidyl transfer, of
the active site tyrosine residue (Tyr-343) in two of the four Flp
monomers. Active site mutants of Flp, in conjunction with suitably
designed recombination substrates, reveal how the active sites for
tyrosine phosphorylation/ dephosphorylation are assembled and
disassembled in space and time, and how the geometry of the DNA
substrate directly influences the path of the reaction. The active site
of Flp provides a model to address the evolutionary design and
optimization of complex active sites. In hybrid DNA-RNA substrates, Flp
shows two types of RNA cleavage activities. One targets the
phosphodiester position involved in DNA recombination, and follows a
related mechanistic path. The second activity targets the immediately
adjacent phosphodiester bond to the 3' side using a distinct mechanism.
The Flp protein, under appropriate conditions, can also be shown to
possess a topoisomerase activity. These results raise pertinent
questions regarding the potential biological significance of the RNA
cleavage activity of a 'nuclease-topoisomerase-recombinase'. They also
have implications for how biological catalysts achieve the balance
between substrate specificity and catalytic versatility.
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