Process for reconstituting the polymerase III* and other subassemblies of E. coli DNA polymerase III holoenzyme from peptide subunits
First Claim
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 andrecovering said polymerase III* subassembly.
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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.
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Citations
53 Claims
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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 andrecovering said polymerase III* subassembly. - View Dependent Claims (2)
- , τ
-
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. - View Dependent Claims (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30)
- , τ
-
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; andrecovering said polymerase III* subassembly. - View Dependent Claims (32, 33, 34)
- , τ
-
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. - View Dependent Claims (36, 37, 38, 39, 40, 41, 42)
- , τ
-
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. - View Dependent Claims (44, 45, 46, 47, 48, 49)
- , τ
-
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. - View Dependent Claims (51)
- , τ
-
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. - View Dependent Claims (53)
- , τ
Specification