Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
1999-03-31
2001-04-24
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S193000
Reexamination Certificate
active
06221642
ABSTRACT:
This work was supported by grants from the National Institute of Health (GM38839) and the National Science Foundation MCB-9303921).
FIELD OF THE INVENTION
This invention relates to a process for reconstituting the polymerase III* and other subassemblies of
E. coli
DNA polymerase III holoenzyme from peptide subunits.
BACKGROUND OF THE INVENTION
The disclosures of all patents and publications cited in the specification are incorporated herein by reference.
DNA polymerase III holoenzyme (“Pol III”) was first purified and determined to be the principal replicase of the
E. coli
chromosome by Kornberg (A. Kornberg, 1982
Supplement to DNA Replication,
Freeman Publications, San Francisco, pp 122-125). The
E. coli
replicase is composed of a DNA polymerase subunit accompanied by multiple accessory proteins and contains at least ten subunits in all (McHenry and Kornberg, 1977,
J. Biol. Chem.,
vol. 252, pp 6478-6484; Maki and Kornberg, 1988,
J. Biol. Chem.,
vol. 263, pp 6551-6559). It has been proposed that chromosomal replicases may contain a dimeric polymerase in order to replicate both the leading and lagging DNA stands concurrently (Sinha et al., 1980,
J. Biol. Chem.,
vol. 225, pp 4290-4303).
One of the features of Pol III which distinguishes it as a chromosomal replicase is its use of ATP to form a tight, gel filterable “initiation complex” on primed DNA (Burgers and Kornberg, 1982,
J. Biol. Chem.,
vol. 257, pp 11468-11473). The holoenzyme initiation complex completely replicates a uniquely primed bacteriophage single-strand DNA (“ssDNA”) genome coated with the ssDNA binding protein (“SSB”), at a speed of at least 500 nucleotides per second at 30° C. without dissociating from an 8.6 kb circular DNA even once (Fay et al., 1981,
J. Biol. Chem.,
vol. 256, pp 976-983; O'Donnell and Kornberg, 1985,
J. Biol. Chem.,
vol. 260, pp 12884-12889; Mok and Marians, 1987,
J Biol. Chem.,
vol. 262, pp 16644-16654). This remarkable processivity, i.e., the high number of nucleotides polymerized in one template binding event, and catalytic speed is in keeping with the rate of replication fork movement in
E. coli,
1 kb/second at 37° C. (Chandler et al., 1975,
J. Mol. Biol.,
vol. 94, pp 127-131).
Within Pol II, the &agr; subunit (dnaE) contains the DNA polymerase activity (Blanar et al., 1984,
Proc. Natl Acad. Sci. USA,
vol. 81, pp 46224626), and the &egr; subunit (dnaQ,mutD) is the proofreading 3′-5′ exonuclease (Scheuetmann and Echols, 1985,
Proc. Natl Acad. Sci. USA,
vol. 81, pp 7747-7751; DeFrancesco et al., 1984,
J. Biol. Chem,
vol. 259, pp 5567-5573). The &agr; subunit forms a tight 1:1 complex with &egr; (Maki and Kornberg, 1985,
J. Biol. Chem.,
vol. 260, pp 12987-12992). Whereas most DNA polymerases have 3′-5′ exonuclease activity, only the holoenzyme relegates this activity to an accessory protein. The following three accessory proteins of the holoenzyme are known to be required for DNA replication as they are products of genes that are essential for cell viability: &bgr; (dnaN) (Burgers et al., 1981,
Proc. Natl Acad. Sci. USA,
vol. 78, pp 5391-5395), &tgr;, and &ggr; (the latter two both encoded by the dnaXZ gene) (Kodaira et al., 1983,
Mol. Gen. Genet.,
vol. 192, pp 80-86).
Important to the assessment of the individual functions of accessory proteins has been the availability of subassemblies of Pol IIl. Subassemblies include Pol III*, which is the holoenzyme lacking only &bgr; (McHenry and Kornberg, 1977,
J. Biol. Chem.
vol. 252, pp 6478-6484); Pol III core, a heterotramer of &agr;&egr;&thgr; that contains the DNA polymerase &agr; subunit and the proofreading 3′5′ exonuclease &egr; subunit (McHenry and Crow, 1979,
J. Biol. Chem.,
vol. 254, pp 1748-1753; Maki and Kornberg, 1985,
J. Biol. Chem.,
vol. 260, pp 12987-12992; Scheuermann and Echols, 1985,
Proc. Natl Acad Sci.,
vol. 81, pp 7747-7751); Pol III′, a dimer of &agr;&egr;&thgr;&tgr; subunits (McHenry, 1982,
J. Biol. Chem.,
vol. 257, pp 2657-2663); the &ggr; complex, &ggr;
2
&dgr;&dgr;′&khgr;&PSgr;, composed of 5 accessory proteins (Maki, and Kornberg, 1988,
J. Biol. Chem.,
vol. 263, pp. 6555-6560); and a &ggr;&khgr;&PSgr; complex (O'Donnell, 1987,
J. Biol. Chem.
vol. 262, pp 16558-16565). Due to the low abundance of the holoenzyme in cells, these subassemblies have hitherto been available only in microgram quantities.
The core polymerase has weak catalytic efficiency and is only processive for approximately 11 nucleotides (Fay et al., 1981,
J. Biol. Chem.,
vol. 256, pp 976-983). The catalytically efficient holoenzyme can be restored upon mixing core with both the &bgr; and the &ggr; complex (Wickner, 1976,
Proc. Natl Acad. Sci. USA,
vol. 73, pp 3511-3515). Reconstitution of the holoenzyme proceeds in two stages. In the first stage, the &ggr; complex and the &bgr; subunit hydrolyze ATP to form a tightly bound “preinitiation complex” clamped onto the primed DNA. In the second stage, the preinitiation complex binds the core and confers onto it highly processive synthesis. ATP is only required in the first stage.
The &ggr; complex both recognizes primed DNA and hydrolyzes ATP to clamp the &bgr; subunit onto DNA. In fact, one &ggr; complex molecule can act catalytically to form many &bgr; clamps on multiple DNA molecules (Stukenberg et al., 1991,
J. Biol. Chem.,
vol. 266, pp 11328-11334). The &ggr; complex therefore has the characteristics of a chaperonin. Namely, it acts catalytically to couple ATP to assembly of a complex (“&bgr;.DNA”). Only the &ggr; and &dgr; subunits are required to clamp &bgr; onto primed DNA (O'Donnell, 1987,
J. Biol. Chem.,
vol. 262, pp 16558-16665).
The holoenzyme Pol III is purified from
E. coli
as a multiprotein particle (O'Donnell, 1992,
Bioessays,
vol. 14, pp 105-111,). The probable orientations of the subunits within the holoenzyme can be deduce from the known interactions among subunits.
Both &ggr; and &tgr; are produced from the same dnaXZ gene. The &ggr; subunit is produced by a frameshift event which occurs after approximately two thirds of the gene has been translated (Tsuchihashi and Kornberg, 1990,
Proc. Natl Acad. Sci. USA,
vol. 87, pp 2516-2520; Flower and McHerry, ibid. pp 3713-3717; Blinkowa and Walker, 1990,
Nuc. Acids Res.,
vol. 18, pp 1725-1729). The frameshift is followed within two amino acids by a stop codon. The &tgr; subunit is the full length product of the dnaXZ gene. Approximately equal amounts of &tgr; and &ggr; are produced in
E. coli
(Kodaira et al., 1993,
Mol. Gen. Genet.,
vol. 192, pp. 80-86).
One of the roles of the &tgr; subunit is to serve as a scaffold to dimerize the polymerase subunits. One indication that &tgr; dimerizes the polymerase is from the purification and characterization of the 4-protein (&agr;&egr;&thgr;&tgr;) subassembly called Pol III′. Pol III′ appears to be a dimer of all four subunits (McHenry, 1982,
J. Biol. Chem.,
vol. 257., pp 2657-2663). Since the &agr;&Egr;&thgr; Pol III core appears to contain only one of each subunit, the dimeric structure of Pol III′ is believed to be due to the &tgr; subunit. Study of pure &agr; and &tgr; subunits has shown the isolated &agr; subunit, i.e., the polymerase is only a monomer, even at high concentration. However, the &tgr; subunit, which is a dimer (Tsuchihashi and Kornberg, 1989,
J. Biol. Chem.,
vol. 264, pp 17790-17795), binds two molecules of &agr;. Hence, &tgr; appears to be the agent of polymerase dimerization. The &tgr; subunit also increases the affinity of the core polymerase for the preinitiation complex (Maki and Kornberg, 1988,
J. Biol. Chem.,
vol. 263, pp. 6561-6569) and is a DNA-dependent ATPase, although the function of its ATPase activity is unknown (Lee and Walker, 1987,
Proc. Natl Acad. Sci. USA,
vol. 84, pp 2713-2717). The &agr;&egr;&thgr; core polymerase appears to form a dimer when it is sufficiently concentrated. Since a 1:1 complex of &agr;&egr; shows no tendency to dimerize to (&agr;&egr;)
2
, the &thgr; subunit has also been proposed to ai
Cornell Research Foundation Inc.
Nixon & Peabody LLP
Weber Jon P.
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