Method for using ref protein as a targeted reca-dependent nuclease

A method of cleaving a duplex DNA molecule at any target defined nucleotide sequence, comprising the steps of:

• (a) assembling a complex of a single-stranded DNA targeting fragment comprising a nucleotide sequence homologous to a target nucleotide sequence of a duplex DNA molecule with a RecA protein comprising:
• (i) the amino acid sequence of SEQ ID NO:1;
• (ii) the amino acid sequence of SEQ ID NO:1 wherein lysine is substituted for glutamic acid at amino acid residue 38, or (iii) an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:1, wherein the RecA functionality is retained;
• (b) contacting the complex that is assembled in step (a) with the duplex DNA molecule;
• and
• (c) contacting a Ref protein comprising:
• (i) SEQ ID NO:2, or (ii) an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:2, wherein the Ref functionality is retained with the duplex DNA molecule;
• wherein both strands of the targeted duplex DNA molecule are cleaved within the sequences defined by homology to the single-stranded DNA used as the targeting fragment.