Process for reconstituting the polymerase III* and other subassemblies of E. coli DNA polymerase III holoenzyme from peptide subunits

• US 5,583,026 A
• Filed: 08/31/1994
• Issued: 12/10/1996
• Est. Priority Date: 08/31/1994
• Status: Expired due to Term

• Current Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Original Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Inventors: O'Donnell, Michael E.
• Primary Examiner(s): Wityshyn, Michael G.
• Assistant Examiner(s): Weber, Jon P.
• Application Number:
  US08/298,945
• Time in Patent Office: 832 Days
• Field of Search: 935/194, 935/193
• US Class Current: 435/194
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Abstract

The process of the invention provides for the reconstitution of the polymerase III* subassembly, Pol III*, of E. coli DNA polymerase III holoenzyme from substantially pure peptide subunits. In the first of two general schemes in which the subunits are added in a specified order, γ and τ are premixed before addition of δ and δ'"'"'. In the second general scheme, δ'"'"' is first assembled onto γ (or τ); then the excess δ'"'"' is removed before adding τ (or γ), following which δ is added. Reconstituted Pol III* had the same subunit composition as purified natural Pol III*, as well as similar activity. Other smaller subassemblies of the polymerase III holoenzyme may also be reconstituted by the process of the invention.

Claims

1. A process for reconstituting in vitro the polymerase III* subassembly of E. coli DNA polymerase III holoenzyme from peptide subunits γ

, τ

, α

, ε

, θ

, δ

, δ

'"'"', χ

, and ψ

, which comprises;

combining said subunits in any order under conditions effective to form said polymerase III* subassembly, provided that γ

is bound with τ

in a single complex prior to incorporating δ

in that complex and

recovering said polymerase III* subassembly.

2. A process of reconstituting polymerase III holoenzyme in vitro which comprises:

reconstituting said polymerase III* subassembly by the process of claim 1;

combining said polymerase III* subassembly with β

subunit under conditions effective to form polymerase III holoenzyme; and

recovering said polymerase III holoenzyme.

3. A process for reconsitituting in vitro the polymerase III* subassembly of E. coli DNA polymerae III holoenzyme from subunits γ

, τ

, α

, ε

, θ

, δ

, δ

'"'"', χ and

ψ

, which comprises;

(a) providing a γ

τ

χ

ψ

complex;

(b) providing an α

ε

θ

core complex;

(c) combining the γ

τ

χ

ψ

complex, the α

ε

θ

core complex, δ

, and δ

'"'"' under conditions effective to form said polymerase III* subassembly; and

(d) recovering said polymerase III* subassembly.

4. A process of reconstituting E. coli DNA polymerase III holoenzyme in vitro, which comprises:

reconstituting said E. coli DNA polymerase III* subassembly by the process of claim 3;

combining said E. coli DNA polymerase III* subassembly with β

subunit under conditions effective to form E. coli DNA polymerase holoenzyme; and

recovering said E. coli DNA polymerase holoenzyme.

5. A process of claim 3, wherein said providing a γ

τ

χ

ψ

complex comprises;

combining γ

, τ

, χ

, and ψ

under conditions effective to form a γ

τ

χ

ψ

complex.

6. A process of claim 5, wherein said combining comprises:

combining γ and

τ

under conditions effective to form a γ

τ

complex; and

combining said γ

τ

complex with χ and

ψ

under conditions effective to form said γ

τ

χ

ψ

complex.

7. A process of claim 5, wherein said combining comprises:

combining γ and

ψ

under conditions effective to form a γ

ψ

complex; and

combining said γ

ψ

complex with τ and

χ

under conditions effective to form said γ

τ

χ

ψ

complex.

8. A process of claim 5, wherein said combining comprises:

combining τ and

ψ

under conditions effective to form a τ

ψ

complex; and

combining said τ

ψ

complex with γ and

χ

under conditions effective to form said γ

τ

χ

ψ

complex.

9. A process of claim 5, wherein said combining comprises:

combining χ

, ψ

, and γ

under conditions effective to form a γ

χ

ψ

complex; and

combining said γ

χ

ψ

complex with τ

under conditions effective to from said γ

τ

χ

ψ

complex.

10. A process of claim 9, wherein said combining χ

, ψ

, and γ

comprises;

forming an aqueous composition (1) comprising subunits χ and

ψ and

incubating said composition (1) under conditions effective to form a composition (1'"'"') comprising χ

ψ

complex and

forming an aqueous composition (3) comprising a first portion of said composition (1'"'"') and γ and

incubating said composition (3) under conditions effective to form a composition (3'"'"') comprising γ

χ

ψ

complex, wherein said combining said γ

χ

ψ

complex with τ

comprises;

forming an aqueous composition (9) comprising said γ

χ

ψ

complex and subunit τ and

incubating said composition (9) under conditions effective to form a composition (9'"'"') comprising γ

τ

χ

ψ

complex, wherein said providing an α

ε

θ

complex comprises;

forming an aqueous composition (6) comprising subunits α

, ε

, and θ and

incubating said composition (6) under conditions effective to form a composition (6'"'"') comprising core complex α

ε

θ

, wherein said combining γ

τ

χ

ψ

complex, the core complex α

ε

θ

, δ

, and δ

'"'"' comprises;

forming an aqueous composition (5) comprising composition (9'"'"') and subunits δ and

δ and

incubating said composition (5) under conditions effective to form a composition (5'"'"') comprising γ

τ

χ

ψ

δ

δ

'"'"' complex;

forming an aqueous composition (7) comprising composition (5'"'"') and composition (6'"'"') and incubating said composition (7) under conditions effective to form a composition (7'"'"') comprising polymerase III* subassembly; and

separating said polymerase III* subassembly from said composition (7'"'"').

11. A process of claim 10, wherein a molar ratio of γ

χ

ψ

complex to τ

of about 1;

1 to 10;

1 is utilized.

12. A process of claim 5, wherein said combining comprises:

combining χ

, ψ

, and τ

under conditions effective to form a τ

χ

ψ

complex; and

combining said τ

χ

ψ

complex with γ

under conditions effective to form said γ

τ

χ

ψ

complex.

13. A process of claim 12, wherein said combining χ

, ψ

, and τ

comprises;

forming an aqueous composition (1) comprising subunits χ and

ψ and

incubating said composition (1) under conditions effective to form a composition (1'"'"') comprising χ

ψ

complex and

forming an aqueous composition (2) comprising a first portion of said composition (1'"'"') and τ and

incubating said composition (2) under conditions effective to form a composition (2'"'"') comprising τ

χ

ψ

complex, wherein said combining said τ

χ

ψ

complex with γ

comprises;

forming an aqueous composition (8) comprising said τ

χ

ψ

complex and subunit γ and

incubating said composition (8) under conditions effective to form a composition (8'"'"') comprising γ

τ

χ

ψ

complex, wherein said providing an α

ε

θ

complex comprises;

forming an aqueous composition (6) comprising subunits α

, ε

, and θ and

incubating said composition (6) under conditions effective to form a composition (6'"'"') comprising core complex α

ε

θ

, wherein said combining γ

τ

χ

ψ

complex, the core complex α

ε

θ

, δ

, and δ

'"'"' comprises;

forming an aqueous composition (5) comprising composition (8'"'"') and subunits δ and

δ

'"'"' and incubating said composition (5) under conditions effective to form a composition (5'"'"') comprising γ

τ

χ

ψ

δ

δ

'"'"' complex;

forming an aqueous composition (7) comprising composition (5'"'"') and composition (6'"'"') and incubating said composition (7) under conditions effective to form a composition (7'"'"') comprising polymerase III* subassembly; and

separating said polymerase III* subassembly from said composition (7'"'"').

14. A process of claim 13, wherein a molar ratio of τ

χ

ψ

complex to γ

of about 0.1;

1 to 1;

1 is utilized.

15. A process of claim 5, wherein said combining comprises:

combining χ and

ψ

under conditions effective to form a χ

ψ

complex; and

combining said χ

ψ

complex with γ and

τ

under conditions effective to form said γ

τ

χ

ψ

complex.

16. A process of claim 15, wherein said combining comprises:

combining said χ

ψ

complex with γ

under conditions effective to form a γ

χ

ψ

complex; and

combining said γ

χ

ψ

complex with τ

under conditions effective to form said γ

τ

χ

ψ

complex.

17. A process of claim 15, wherein said combining comprises:

combining said χ

ψ

complex with τ

under conditions effective to form a τ

χ

ψ

complex; and

combining said τ

χ

ψ

complex with γ

under conditions effective to form said γ

τ

χ

ψ

complex.

18. A process of claim 5, wherein said combining comprises:

combining χ

, ψ

, and γ

under conditions effective to form a γ

χ

ψ

complex; and

combining χ

, ψ

, and τ

under conditions effective to form a τ

χ

ψ

complex; and

combining said γ

χ

ψ

complex and said τ

χ

ψ

complex under conditions effective to form said γ

τ

χ

ψ

complex.

19. A process of claim 18, wherein said combining χ

, ψ

, and τ and

said combining χ

, ψ

, and γ

comprises;

forming an aqueous composition (1) comprising subunits χ and

ψ and

incubating said composition (1) under conditions effective to form a composition (1'"'"') comprising χ

ψ

complex;

forming an aqueous composition (2) comprising a first portion of said composition (1'"'"') and subunit τ and

incubating said composition (2) under conditions effective to form a composition (2'"'"') comprising τ

χ

ψ

complex; and

forming an aqueous composition (3) comprising a second portion of said composition (1'"'"') and subunit γ and

incubating said composition (3) under conditions effective to form a composition (3'"'"') comprising γ

χ

ψ

complex, wherein said combining said γ

χ

ψ

complex and said τ

χ

ψ

complex comprises;

forming an aqueous composition (4) comprising said compositions (2'"'"') and (3'"'"') and incubating said composition (4) under conditions effective to form a composition (4'"'"') comprising γ

τ

χ

ψ

complex, wherein said providing an α

ε

θ

core complex comprises;

forming an aqueous composition (6) comprising subunits α

, ε

, and θ and

incubating said composition (6) under conditions effective to form a composition (6'"'"') comprising core complex α

ε

θ

, wherein said combining the γ

τ

χ

ψ

complex, the α

ε

θ

core complex, δ

, and δ

'"'"' comprises;

forming an aqueous composition (5) comprising said composition (4'"'"') and subunit δ and

δ

'"'"' and incubating said composition (5) under conditions effective to form a composition (5'"'"');

forming an aqueous composition (7) comprising said compositions (5'"'"') and (6'"'"') and incubating said composition (7) under conditions effective to form a composition (7'"'"') comprising the polymerase III* subassembly; and

separating said polymerase III* subassembly from said composition (7'"'"').

20. A process of claim 19, wherein each of said incubating steps is carried out for a time period of about 5 minutes to 120 minutes and at a temperature of about 0°

C. to 45°

C.

21. A process of claim 20, wherein said time period is about 15 minutes to 60 minutes and said temperature is about 10°

C. to 20°

C.

22. A process of claim 19, wherein a molar ratio of γ

to τ

of about 1;

1 to 5;

1 and molar ratios of χ

to τ and

ψ

to τ

each of about 2;

1 to 10;

1 are utilized.

23. A process of claim 22, wherein the molar ratio of γ

to τ

is about 1.5;

1 to 3;

1, the molar ratio of χ

to τ

is about 4;

1 to 7;

1, and the molar ratio of ψ

to τ

is about 4;

1 to 7;

1.

24. A process of claim 19, wherein molar ratios of δ

to τ and

δ

'"'"' to τ

each of about 2;

1 to 10;

1 are utilized.

25. A process of claim 24, wherein the molar ratio of δ

to τ

is about 4;

1 to 6;

1 and the molar ratio of δ

'"'"' to τ

is about 4;

1 to 6;

1.

26. A process of claim 19, wherein a molar ratio of α

to τ

of about 1;

1 to 5;

1 and molar ratios of ε

to τ and

θ

to τ

each of about 2;

1 to 10;

1 are utilized.

27. A process of claim 26, wherein the molar ratio of α

to τ

is about 2;

1 to 3;

1, the molar ratio of ε

to τ

is about 3;

1 to 5;

1, and the molar ratio of θ

to τ

is about 4;

1 to 7;

1.

28. A process of claim 3, wherein said providing an α

ε

θ

core complex comprises;

combining α

, ε

, and θ

under conditions effective to form an α

ε

θ

core complex.

29. A process of claim 3, wherein said combining comprises:

combining said γ

τ

χ

ψ

complex with δ and

δ

'"'"' under conditions effective to form a γ

τ

χ

ψ

δ

δ

'"'"' complex.

30. A process of claim 29, wherein said combining comprises:

combining said γ

τ

χ

ψ

δ

δ

'"'"' complex with said α

ε

θ

core complex under conditions effective to form said polymerase III* subassembly.

31. A process for reconstituting in vitro the polymerase III* subassembly of E. coli DNA polymerase III holoenzyme from the substantially pure peptide subunits γ

, τ

, α

, ε

, θ

, δ

, δ

'"'"', χ

, and ψ

, which comprises;

combining γ

, ψ

, and δ

'"'"' or τ

, ψ

, and δ

'"'"' under conditions effective to form, respectively, substantially pure γ

ψ

δ

'"'"' complex or substantially pure τ

ψ

δ

'"'"' complex;

combining said γ

χ

δ

'"'"' complex with τ

or said τ

ψ

δ

'"'"' complex with γ

under conditions effective to form γ

τ

ψ

δ

'"'"' complex;

combining said γ

τ

ψ

δ

'"'"' complex with α

, ε

, θ

, δ

, and χ

in any order under conditions effective to form said polymerase III* subassembly; and

recovering said polymerase III* subassembly.

32. A process of claim 31, wherein said combining said γ

τ

ψ

δ

'"'"' complex with α

, ε

, θ

, δ

, and χ

comprises;

combining χ and

δ

with said γ

τ

ψ

δ

'"'"' complex under conditions effective to form a γ

τ

χ

ψ

δ

δ

'"'"' complex; and

combining said γ

τ

χ

ψ

δ

δ

'"'"' complex with α

, ε

, and θ

under conditions effective to form said polymerase III* subassembly.

33. A process of claim 32, wherein said combining said γ

τ

χ

ψ

δ

δ

'"'"' complex with α

, ε

, and θ

further comprises;

combining α

, ε

, and θ

under conditions effective to form an α

ε

θ

core complex; and

combining said α

ε

θ

core complex with said γ

τ

χ

ψ

δ

δ

'"'"' complex under conditions effective to form said polymerase III* subassembly.

34. A process of reconstituting polymerase III holoenzyme in vitro which comprises:

reconstituting said polymerase III* subassembly by the process of claim 31;

combining said polymerase III* subassembly with β

subunit under conditions effective to form polymerase III holoenzyme; and

recovering said polymerase III holoenzyme.

35. A process for reconstituting in vitro the polymerase III* subassembly of E. coli DNA polymerase III holoenzyme from subunits γ

, τ

, α

, ε

, θ

, δ

, δ

'"'"', χ and

ψ

, which comprises;

(a) providing a γ

χ

ψ

δ

'"'"' complex;

(b) providing an α

ε

θ

core complex;

(c) combining said γ

χ

ψ

δ

'"'"' complex with τ

under conditions effective to form a γ

τ

χ

ψ

δ

'"'"' complex;

(d) combining said γ

τ

χ

ψ

δ

'"'"' complex with δ

under conditions effective to form a γ

τ

χ

ψ

δ

δ

'"'"' complex;

(e) combining said γ

τ

χ

ψ

δ

δ

'"'"' complex and said α

ε

θ

core complex under conditions effective to form said polymerase III* subassembly; and

(f) recovering said polymerase III* subassembly.

36. A process of claim 35, wherein said providing a γ

χ

ψ

δ

'"'"' complex comprises;

combining χ and

ψ

under conditions effective to form a χ

ψ

complex; and

combining said χ

ψ

complex with γ and

δ

'"'"' under conditions effective to form a γ

χ

ψ

δ

'"'"' complex.

37. A process of claim 35, wherein said combining said γ

χ

ψ

δ

'"'"' complex with τ

comprises;

combining said γ

χ

ψ

δ

'"'"' complex with τ

under conditions effective to form γ

τ

χ

ψ

δ

'"'"' complex, and then combining said γ

τ

χ

ψ

δ

'"'"' complex with δ

under conditions effective to form γ

τ

χ

ψ

δ

δ

'"'"' complex.

38. A process of claim 35, wherein said providing a γ

χ

ψ

δ

'"'"' complex comprises;

forming an aqueous composition (1) comprising subunits χ and

ψ and

incubating said composition (1) under conditions effective to form a composition (1'"'"') comprising χ

ψ

complex;

forming an aqueous composition (10) comprising said composition (1'"'"') and subunits δ

'"'"', and γ

, and incubating said composition (10) under conditions effective to form a composition (10'"'"') comprising γ

χ

ψ

δ

'"'"' complex; and

separating γ

χ

ψ

δ

'"'"' complex from said composition (10'"'"'), wherein said combining said γ

χ

ψ

δ

'"'"' complex with τ

comprises;

forming an aqueous composition (11) comprising said composition (10'"'"') and subunit τ and

incubating said composition (11) under conditions effective to form a composition (11'"'"') comprising γ

τ

χ

ψ

δ

'"'"' complex, wherein said combining said γ

τ

χ

ψ

δ

'"'"' complex with δ

comprises;

forming an aqueous composition (12) comprising said composition (11'"'"') and subunit δ and

incubating said composition (12) under conditions effective to form a composition (5'"'"') comprising γ

τ

χ

ψ

δ

δ

'"'"' complex, wherein said providing an α

ε

θ

core complex comprises;

forming an aqueous composition (6) comprising subunits α

, ε

, and θ and

incubating said composition (6) under conditions effective to form a composition (6'"'"') comprising core complex α

ε

θ

, wherein said combining said γ

τ

χ

ψ

δ

δ

'"'"' complex comprises;

forming an aqueous composition (7) comprising said compositions (5'"'"') and (6'"'"') and incubating said composition (7) under conditions effective to form a composition (7'"'"') comprising the polymerase III* subassembly; and

separating said polymerase III* subassembly from said composition (7'"'"').

39. A process of reconstituting polymerase III holoenzyme in vitro which comprises:

reconstituting said polymerase III* subassembly by the process of claim 35;

combining said polymerase III* subassembly with β

subunit under conditions effective to form polymerase III holoenzyme; and

recovering said polymerase III holoenzyme.

40. A process of claim 38, wherein each of said incubating steps is carried out for a time period of about 5 minutes to 120 minutes and at a temperature of about 0°

C. to 45°

C.

41. A process of claim 40, wherein said time period is about 15 minutes to 60 minutes and said temperature is about 10°

to 20°

C.

42. A process of claim 38, wherein a molar ratio of γ

χ

ψ

δ

'"'"' complex to τ

of about 1;

1 to 10;

1 is utilized.

43. A process for reconstituting in vitro the polymerase III* subassembly of E. coli DNA polymerase III holoenzyme from subunits γ

, τ

, α

, ε

, θ

, δ

, δ

'"'"', χ and

ψ

, which comprises;

(a) providing a τ

χ

ψ

δ

'"'"' complex;

(b) providing an α

ε

θ

core complex;

(c) combining said τ

χ

ψ

δ

'"'"' complex with γ

under conditions effective to form a γ

τ

χ

ψ

δ

'"'"' complex;

(d) combining said γ

τ

χ

ψ

δ

'"'"' complex with δ

under conditions effective to form a γ

τ

χ

ψ

δ

δ

'"'"' complex;

(e) combining said γ

τ

χ

ψ

δ

δ

'"'"' complex with said α

ε

θ

core complex under conditions effective to form said polymerase III* subassembly; and

(f) recovering said polymerase III* subassembly.

44. A process of claim 43, wherein said providing a τ

χ

ψ

δ

'"'"' complex comprises;

combining χ and

ψ

under conditions effective to form a χ

ψ

complex; and

combining said χ

ψ

complex with τ and

δ

'"'"' under conditions effective to form a τ

χ

ψ

δ

'"'"' complex.

45. A process of claim 43, wherein said providing a τ

χ

ψ

δ

'"'"' complex comprises;

forming an aqueous composition (1) comprising subunits χ and

ψ and

incubating said composition (1) under conditions effective to form a composition (1'"'"') comprising χ

ψ

complex;

forming an aqueous composition (13) comprising said composition (1'"'"') and subunits δ

'"'"' and τ

, and incubating said composition (13) under conditions effective to form a composition (13'"'"') comprising τ

χ

ψ

δ

'"'"' complex; and

separating τ

χ

ψ

δ

'"'"' complex from said composition (13'"'"'), wherein said combining said τ

χ

ψ

δ

'"'"' complex with γ

comprises;

forming an aqueous composition (14) comprising said composition (13'"'"') and subunit γ and

incubating said composition (14) under conditions effective to form a composition (14'"'"') comprising γ

τ

χ

ψ

δ

'"'"' complex, wherein said combining said γ

τ

χ

ψ

δ

'"'"' complex with δ

comprises;

forming an aqueous composition (15) comprising said composition (14'"'"') and subunit δ and

incubating said composition (15) under conditions effective to form a composition (5'"'"') comprising γ

τ

χ

ψ

δ

δ

'"'"' complex, wherein said providing an α

ε

θ

core complex comprises;

forming an aqueous composition (6) comprising subunits α

, ε

, and θ and

incubating said composition (6) under conditions effective to form a composition (6'"'"') comprising core complex α

ε

θ

, wherein said combining said γ

τ

χ

ψ

δ

δ

'"'"' complex comprises;

forming an aqueous composition (7) comprising said compositions (5'"'"') and (6'"'"') and incubating said composition (7) under conditions effective to form a composition (7'"'"') comprising the polymerase III* subassembly; and

separating said polymerase III* subassembly from said composition (7'"'"').

46. A process of reconstituting polymerase III holoenzyme in vitro which comprises:

reconstituting said polymerase III* subassembly by the process of claim 43;

combining said polymerase III* subassembly with β

subunit under conditions effective to form polymerase III holoenzyme; and

recovering said polymerase III holoenzyme.

47. A process of claim 45, wherein each of said incubating steps is carried out for a time period of about 5 minutes to 120 minutes and at a temperature of about 0°

C. to about 45°

C.

48. A process of claim 47, wherein said time period is about 15 minutes to 60 minutes and said temperature is about 10°

C. to 20°

C.

49. A process of claim 45, wherein a molar ratio of τ

χ

ψ

δ

'"'"' complex to γ

of about 0.1;

1 to 1;

1 is utilized.

50. A process for reconstituting in vitro the polymerase III* subassembly of E. coli DNA polymerase III holoenzyme from subunits γ

, τ

, α

, ε

, θ

, δ

, δ

'"'"', χ and

ψ

, which comprises;

(a) providing a γ

ψ

δ

'"'"' complex;

(b) combining said γ

ψ

δ

'"'"' complex with τ

under conditions effective to form γ

τ

ψ

δ

'"'"' complex;

(c) combining said γ

τ

ψ

δ

'"'"' complex with α

, ε

, θ

, δ

, and χ

in any order under conditions effective to form said polymerase III* subassembly; and

(d) recovering said polymerase III* subassembly.

51. A process of reconstituting polymerase III holoenzyme in vitro which comprises:

reconstituting said polymerase III* subassembly by the process of claim 50;

combining said polymerase III* subassembly with subunit under conditions effective to form polymerase III holoenzyme; and

recovering said polymerase III holoenzyme.

52. A process for reconstituting in vitro the polymerase III* subassembly of E. coli DNA polymerase III holoenzyme from subunits γ

, τ

, α

, ε

, θ

, δ

, δ

'"'"', χ and

ψ

, which comprises;

(a) providing a τ

ψ

δ

'"'"' complex;

(b) combining said τ

ψ

δ

'"'"' complex with γ

under conditions effective to form γ

τ

ψ

δ

'"'"' complex;

(c) combining said γ

τ

ψ

δ

'"'"' complex with α

, ε

, θ

, δ

, and χ

in any order under conditions effective to form said polymerase III* subassembly; and

(d) recovering said polymerase III* subassembly.

53. A process of reconstituting polymerase III holoenzyme in vitro which comprises:

reconstituting said polymerase III* subassembly by the process of claim 52;

combining said polymerase III* subassembly with β

subunit under conditions effective to form polymerase III holoenzyme; and

recovering said polymerase III holoenzyme.