Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
2001-03-28
2004-01-13
Hutson, Richard (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S183000, C435S195000, C435S006120, C530S350000, C536S023100, C536S023200, C536S023700
Reexamination Certificate
active
06677146
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to gene and amino acid sequences encoding DNA polymerase III holoenzyme subunits and structural genes from thermophilic organisms. In particular, the present invention provides DNA polymerase III holoenzyme subunits and accessory proteins of
T. thermophilus.
The present invention also provides antibodies and other reagents useful to identify DNA Polymerase III molecules.
2. Background Art
Bacterial cells contain three types of DNA polymerases termed polymerase I, II and III. DNA polymerase III (pol III) is responsible for the replication of the majority of the chromosome. Pol III is referred to as a replicative polymerase; replicative polymerases are rapid and highly processive enzymes. Pol I and II are referred to as non-replicative polymerases although both enzymes appear to have roles in replication. DNA polymerase I is the most abundant polymerase and is responsible for some types of DNA repair, including a repair-like reaction that permits the joining of Okazaki fragments during DNA replication. Pol I is essential for the repair of DNA damage induced by UV irradiation and radiomimetic drugs. Pol II is thought to play a role in repairing DNA damage which induces the SOS response and in mutants which lack both pol I and III, pol II repairs UV-induced lesions. Pol I and II are monomeric polymerases while pol III comprises a multisubunit complex.
In
E. coli,
pol III comprises the catalytic core of the
E. coli
replicase. In
E. coli,
there are approximately 400 copies of DNA polymerase I per cell, but only 10-20 copies of pol III (Komberg and Baker,
DNA Replication,
2d ed., W. H. Freeman & Company, [1992], pp. 167; and Wu et al. J. Biol. Chem., 259:12117-12122 [1984]). The low abundance of pol III and its relatively feeble activity on gapped DNA templates typically used as a general replication assays delayed its discovery until the availability of mutants defective in DNA polymerase I (Kornberg and Gefter, J. Biol. Chem., 47:5369-5375 [1972]).
The catalytic subunit of pol III is distinguished as a component of
E. coli
major replicative complex, apparently not by its intrinsic catalytic activity, but by its ability to interact with other replication proteins at the fork. These interactions confer upon the enzyme enormous processivity. Once the DNA polymerase III holoenzyme associates with primed DNA, it does not dissociate for over 40 minutes—the time required for the synthesis of the entire 4 Mb
E. coli
chromosome (McHenry, Ann. Rev. Biochem., 57:519-550 [1988]). Studies in coupled rolling circle models of the replication fork suggest the enzyme can synthesize DNA 150 kb or longer without dissociation in vitro (Mok and Marians, J. Biol. Chem., 262:16644-16654 [1987]; Wu et al., J. Biol. Chem., 267:4030-4044 [1992]). The essential interaction required for this high processivity is an interaction between the &agr; catalytic subunit and a dimer of &bgr;, a sliding clamp processivity factor that encircles the DNA template like a bracelet, permitting it to rapidly slide along with the associated polymerase, but preventing it from falling off (LaDuca et al., J. Biol. Chem., 261:7550-7557 [1986]; Kong et al., Cell 69:425-437 [1992]). The &bgr;-&agr; association apparently retains the polymerase on the template during transient thermal fluctuations when it might otherwise dissociate.
The &bgr;
2
bracelet cannot spontaneously associate with high molecular weight DNA, it requires a multiprotein DnaX-complex to open and close it around DNA using the energy of ATP hydrolysis (Wickner, Proc. Natl. Acad. Sci. USA 73:35411-3515 [1976]; Naktinis et al., J. Biol. Chem., 270:13358-13365 [1985]; and Dallmann et al., J. Biol. Chem., 270:29555-29562 [1995]). In
E. coli,
the dnaX gene encodes two proteins, &tgr; and &ggr;. &ggr; is generated by a programmed ribosomal frameshifting mechanism five-sevenths of the way through dnaX mRNA, placing the ribosome in a −1 reading frame where it immediately encounters a stop codon (Flower and McHenry Proc. Natl. Acad. Sci. USA 87:3713-3717 [1990]; Blinkowa and Walker, Nucl. Acids Res., 18:1725-1729 [1990]; and Tsuchihashi and Kornberg, Proc. Nati. Acad. Sci. USA 87:2516-2520 [1990]). In
E. coli,
the DnaX-complex has the stoichiometry &ggr;
2
&tgr;
2
&dgr;
1
&dgr;′
1
&khgr;
1
&igr;
1
(Dallmann and McHenry, J. Biol. Chem., 270:29563-29569 [1995]). The &tgr; protein contains an additional carboxyl-terminal domain that interacts tightly with the polymerase, holding two polymerases together in one complex that can coordinately replicate the leading and lagging strand of the replication fork simultaneously (McHenry, J. Biol. Chem., 257:2657-2663 [1982]; Studwell and O'Donnell, Biol. Chem., 266:19833-19841 [1991]; McHenry, Ann. Rev. Biochem. 57:519-550 [1988]).
Conservation of a frameshifting mechanism to generate related ATPases is significant in that, by analogy to
E. coli,
can both assemble a processivity factor onto primed DNA. In
E. coli,
ribosomes frameshift at the sequence A AAA AAG into a −1 frame where the lysine UUU anticodon tRNA can base pair with 6As before elongating (Flower and McHenry, Proc. Natl. Acad. Sci. USA 87:3713-3717 [1990]; Blinkowa and Walker, Nucl. Acids Res., 18:1725-1729 [1990]; and Tsuchihashi and Kornberg, Proc. Natl. Acad. Sci. USA 87:2516-2520 [1990]).
Pol IIIs are apparently conserved throughout mesophilic eubacteria. In addition to
E. coli
and related proteobacteria, the enzyme has been purified from the firmicute
Bacillus subtilis
(Low et al., J. Biol. Chem., 251:1311-1325 [1976]; Hammond and Brown [1992]). With the proliferation of bacterial genomes sequenced, by inference from DNA sequence, pol III exists in organisms as widely divergent as Caulobacter, Mycobacteria, Mycoplasma,
B. subtilis
and Synechocystis. The existence of dnaX and dnaN (structural gene for &bgr;) is also apparent in these organisms. These general replication mechanisms are conserved even more broadly in biology. Although eukaryotes do not contain polymerases homologous to pol III, eukaryotes contain special polymerases devoted to chromosomal replication and &bgr;-like processivity factors (PCNA) and DnaX-like ATPases (RFC, Activator I) that assemble these processivity factors on DNA (Yoder and Burgers, J. Biol. Chem., 266:22689-22697 [1991]; Brush and Stillman, Meth. Enzymol., 262:522-548 [1995]; Uhlmann et al., Proc. Nati. Acad. Sci. USA 93:6521-6526 [1996]).
Helicases serve a variety of functions in DNA metabolism. Cellular (
E. coli
dnaB, priA, and rep proteins), phage (T4 gene 41 and dda proteins; T7 gene 4 protein), and viral (SV40 T antigen; HSV-1 UL5/UL52 complex and UL9 protein) helicases are involved in the initiation of replication, by unwinding DNA so that other proteins of the replication complex can assemble on the ssDNA. These proteins also participate in the elongation phase of replication, by unwinding the duplex DNA ahead of this complex to provide the required template. Other helicases (e.g., the
E. coli
recBCD and recQ proteins) are implicated in recombination by genetic criteria. Another class of helicases includes the
E. coli
uvrAB and uvrD. These helicases act in nucleotide excision repair or methyl-directed mismatch repair during both pre-incision (recognition of DNA damage or alteration) and post-incision (displacement of damaged fragment) steps. See, for example, U.S. Pat. No. 5,747,247.
DNA mispairing can occur in vivo and is recognized and corrected by repair proteins. Mismatch repair has been studied most intensively in
E. coli, Salmonella typhimurium,
and
S. pneumoniae.
The MutS, MutH and MutL proteins of
E. coli
are involved in the repair of DNA mismatches, as is the product of the uvrD gene in
E. coli,
helicase II. See, for
Bullard James M.
Janjic Nebojsa
Kery Vladimir
McHenry Charles S.
Hutson Richard
Replidyne, Inc.
Swanson & Bratschun LLC
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