Nucleic acid modifying enzymes
This invention provides for an improved generation of novel nucleic acid modifying enzymes. The improvement is the fusion of a sequence-non-specific nucleic-acid-binding domain to the enzyme in a manner that enhances the ability of the enzyme to bind and catalytically modify the nucleic acid.
1. A protein comprising two joined heterologous domains:
a sequence non-specific double-stranded nucleic acid binding domain that
comprises an amino acid sequence that has at least 75% sequence identity to SEQ ID NO:2; and
a DNA polymerase domain
wherein the presence of the sequence non-specific double-stranded nucleic acid binding domain enhances the processivity of the polymerase domain compared to an identical protein that does not have the sequence non-specific double-stranded nucleic acid binding domain joined thereto.
2. The protein of claim 1 , wherein the sequence non-specific double-stranded nucleic acid binding domain and the DNA polymerase domain are covalently linked.
3. The protein of claim 1 , wherein the DNA polymerase domain has thermally stable polymerase activity.
4. The protein of claim 1 , wherein the DNA polymerase domain comprises a family A polymerase domain.
5. The protein of claim 4 , wherein the family A polymerase domain is a Thermus polymerase domain.
6. The protein of claim 4 , wherein the family A polymerase domain is a Taq polymerase domain.
7. The protein of claim 3 , wherein the DNA polymerase domain is a ΔTaq domain.
8. The protein of claim 1 , wherein the polymerase domain is a family B polymerase domain.
9. The protein of claim 8 , wherein the family B polymerase domain is a Pyrococcus DNA polymerase I domain.
10. The protein of claim 9 , wherein the Pyrococcus polymerase domain is a Pyrococcus furiosus domain.
11. A protein comprising two joined heterologous domains:
a sequence non-specific double-stranded nucleic acid binding domain that comprises an amino acid sequence that has at least 75% sequence identity to the Sac7d sequence set forth in amino acids 7-71 of SEQ ID NO:10; and
a DNA polymerase domain,
wherein the presence of the sequence non-specific double-stranded nucleic acid binding domain enhances the processivity of the polymerase domain compared to an identical protein that does not have the sequence non-specific double-stranded nucleic acid binding domain joined thereto.
12. The protein of claim 11 , wherein the sequence non-specific double-stranded nucleic acid binding domain and the DNA polymerase domain are covalently linked.
13. The protein of claim 11 , wherein the sequence non-specific double-stranded nucleic acid binding domain comprises an amino acid sequence that has at least 90% sequence identity to the Sac 7d sequence set forth in SEQ ID NO:10.
14. The protein of claim 11 , wherein the DNA polymerase domain has thermally stable polymerase activity.
15. The protein of claim 11 , wherein the DNA polymerase domain comprises a family A polymerase domain.
16. The protein of claim 14 , wherein the DNA polymerase domain is a Thermus polymerase domain.
17. The protein of claim 16 , wherein the Thermus polymerase domain is a Taq polymerase domain.
18. The protein of claim 14 , wherein the DNA polymerase domain is a ΔTaq domain.
19. The protein of claim 11 , wherein the polymerase domain is a family B polymerase domain.
20. The protein of claim 19 , wherein the family B polymerase domain is a Pyrococcus DNA polymerase I domain.
21. The protein of claim 20 , wherein the Pyrococcus polymerase domain is a Pyrococcus furiosus domain.
22. The protein of claim 1 , wherein the sequence non-specific double-stranded nucleic acid binding domain comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:2.
23. The protein of claim 11 , wherein the sequence non-specific double-stranded nucleic acid binding domain comprises an amino acid sequence that has at least 85% sequence identity to the Sac 7d sequence set forth in SEQ ID NO:10.