RecA mutants
The present invention provides RecA mutant proteins, having either a single mutation or a double mutation. The RecA mutant proteins are highly proficient in both SSB displacement and steady state binding of DNA in the presence or absence of SSB as compared to the wild-type protein. The single RecA mutant, RecAΔC17, has 17 amino acid residues removed from the carboxyl terminus. The double mutant RecA, RecAΔC17/E38K, combines the 17 amino acid residue C-terminal deletion of RecAΔC17, with a single amino acid change from Glutamate to Lysine at position 38. These RecA mutant proteins are pH sensitive allowing control over formation of products. Hence, methods of using the novel RecA mutants and kits having the RecA mutants as components thereof are also contemplated by the present invention.
1. A isolated double mutant Escherichia Coli RecA protein comprising a truncation of from 13 to 25 amino acid residues; from the carboxyl terminus and an amino acid change from a glutamate to a basic amino acid at position 38.
2. The protein of claim 1 wherein the truncation is 17 amino acid residues from the carboxyl terminus.
3. The protein of claim 1 wherein the basic amino acid is lysine.
4. The protein of claim 1 wherein the basic amino acid is to arginine.
5. The protein of claim 1 wherein 17 amino acid residues are truncated from the carboxyl terminus and the glutamate is changed to lysine, as set forth in SEQ ID NO. 3.
6. The protein of claim 5 comprising an enhanced capacity to displace a DNA binding protein as compared to wild-type RecA.
7. The protein of claim 6 wherein the protein is a single stranded DNA binding protein, SSB.
8. The protein of claim 5 comprising an increased steady-state DNA binding capacity during a DNA strand exchange reaction as compared to wild-type RecA.
9. The protein of claim 8 wherein the DNA is single-stranded.
10. The protein of claim 8 wherein the DNA is double-stranded.
11. The protein of claim 10 wherein the double-stranded DNA is linear or circular.
12. The protein of claim 8 wherein the DNA strand exchange reaction is pH dependent.
13. The protein of claim 12 wherein the DNA strand exchange reaction induces complete product formation between a pH of 8.0 to 9.0.
14. The protein of claim 12 wherein the DNA strand exchange reaction induces complete product formation at a pH of 8.5.
15. The protein of claim 8 wherein the DNA strand exchange reaction is Mg2+ concentration dependent.
16. The protein of claim 15 wherein the Mg2+ concentration is between 4 mM–8 mM.
17. The protein of claim 15 wherein the Mg2+ concentration is 5 mM.
18. The protein of claim 5 wherein the protein promotes an extended reaction, wherein the extended reaction is at least a three-strand exchange reaction.
19. A kit comprising the protein of claim 1 .
20. A kit comprising the protein of claim 5 .
21. An isolated double mutant E. Coli RecA protein comprising; a truncation of up to 17 amino acid residues from the carboxyl terminus and an amino acid change from a glutamate to a basic amino acid at position 38.
22. A kit comprising the protein of claim 21 .
23. An isolated double mutant E. Coli RecA protein comprising a truncation of from 13 to 20 amino acid residues; from the carboxyl terminus and an amino acid change from a glutamate to a basic amino acid at position 38.
24. An isolated double mutant E. Coli RecA protein comprising a truncation of from 13 to 17 amino acid residues; from the carboxyl terminus and an amino acid change from a glutamate to a basic amino acid at position 38.