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 nucleic acid encoding a polypeptide 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; or 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 polymerase domain that does not have the sequence non-specific double-stranded nucleic acid binding domain joined thereto.
2. The nucleic acid of claim 1 , wherein the sequence non-specific double-stranded nucleic acid binding domain comprises an amino acid sequence that has at least 75% sequence identity to SEQ ID NO:2.
3. The nucleic acid of claim 2 , wherein the sequence non-specific double-stranded nucleic acid binding domain comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:2.
4. The nucleic acid of claim 3 , wherein the sequence non-specific double-stranded nucleic acid binding domain comprises the amino acid sequence of SEQ ID NO:2.
5. The nucleic acid of claim 2 , wherein the DNA polymerase domain has thermally stable polymerase activity.
6. The nucleic acid of claim 2 , wherein the DNA polymerase domain is a family A polymerase domain.
7. The nucleic acid of claim 5 , wherein DNA polymerase domain is a Thermus polymerase domain.
8. The nucleic acid of claim 5 , wherein the DNA polymerase domain is a Taq polymerase domain.
9. The nucleic acid of claim 5 , wherein the DNA polymerase domain is a ΔTaq polymerase domain.
10. The nucleic acid of claim 2 , wherein the DNA polymerase domain is a family B polymerase domain.
11. The nucleic acid of claim 5 , wherein the DNA polymerase domain is a Pyrococcus DNA polymerase domain.
12. The nucleic acid of claim 5 , wherein the DNA polymerase domain is a Pyrococcus furiosus polymerase domain.
13. The nucleic acid of claim 1 , wherein the sequence non-specific double-stranded nucleic acid binding domain 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.
14. The nucleic acid of claim 13 , 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 Sac7d sequence set forth in amino acids 7-71 of SEQ ID NO:10.
15. The nucleic acid of claim 13 , wherein the sequence non-specific double-stranded nucleic acid binding domain comprises the amino acid sequence of the Sac7d sequence set forth in amino acids 7-71 of SEQ ID NO:10.
16. The nucleic acid of claim 13 , wherein the DNA polymerase domain has thermally stable polymerase activity.
17. The nucleic acid of claim 13 , wherein the DNA polymerase domain is a family A polymerase domain.
18. The nucleic acid of claim 16 , wherein the DNA polymerase domain is a Thermus polymerase domain.
19. The nucleic acid of claim 16 , wherein the DNA polymerase domain is a Taq polymerase domain.
20. The nucleic acid of claim 16 , wherein the DNA polymerase domain is a ΔTaq domain.
21. The nucleic acid of claim 13 , wherein the DNA polymerase domain is a family B polymerase domain.
22. The nucleic acid of claim 16 , wherein the DNA polymerase domain is a Pyrococcus domain.
23. The nucleic acid of claim 16 , wherein the DNA polymerase domain is a Pyrococcus furiosus domain.
24. An expression vector comprising a nucleic acid of claim 1 .
25. A host cell comprising a nucleic acid of claim 1 .
26. A method of producing a polypeptide comprising two joined heterologous domains, wherein the method comprises culturing a host cell of claim 25 under conditions in which the polypeptide comprising the two joined heterologous domains is expressed.
27. The method of claim 26 , further comprising purifying the polypeptide produced by the host cell.