Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-08-28
2001-12-25
Zitomer, Stephanie W. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S091200, C530S350000, C536S023100
Reexamination Certificate
active
06333153
ABSTRACT:
FIELD OF THE INVENTION
The field of the invention is DNA mismatch protein binding, including animals useful as models for tumorigenesis, apoptosis, and aging.
BACKGROUND OF THE INVENTION
DNA Mismatch Repair
The most widely accepted model for DNA post-replication mismatch repair is based largely on the model of the DNA adenine methylation (Dam)-Instructed pathway of
Escherichia coli
proposed by Modrich (1986, Basic Life Sci. 38:303-310; Modrich, 1987, Ann. Rev. Biochem. 56:435-466; Modrich, 1989, J. Biol. Chem. 264:6597-6600; Modrich, 1991, Annu. Rev. Genet. 25:229-253; Modrich et al., 1996, Annu. Rev. Biochem. 65:101-133). According to this model, the MutS protein recognizes and binds mismatched nucleotides resulting from polymerase misincorporation errors to form a MutS-DNA product (Su et al., 1986, Proc. Natl. Acad. Sci., USA 83:5057-5061; Su et al., 1988, J. Biol. Chem. 263:6829-6835). MutS mismatch binding is followed by the interaction of MutL protein with the MutS-DNA product (Grilley et al., 1990, Mutat. Res. 236:253-267), which accelerates ATP-dependent translocation of the MutS-MutL complex (Allen et al., 1997, EMBO J. 16:4467-4476) to a hemimethylated GATC Dam site to which MutH protein is bound (Welsh et al., 1987, J. Biol. Chem. 262:15624-15629; Au et al., 1992, J. Biol. Chem. 267:12142-12148). The MutS-MutL complex stimulates an intrinsic endonuclease activity of MutH protein, which cleaves the non-methylated (i.e. more recently replicated) DNA strand (Welsh et al., 1987, J. Biol. Chem. 262:15624-15629; Lahue et al., 1987, Proc. Natl. Acad. Sci. USA 84:1482-1486; Su et al., 1989, Genome 31:104-111; Cooper et al., 1993, J. Biol. Chem. 268:11823-11829; Grilley et al., 1993, J. Biol. Chem. 268:11830-11837). Strand cleavage enables one of three single-stranded exonucleases of
E. coli
(RecJ, Exol, ExoVII) to degrade the non-methylated strand, which can then be re-synthesized by the
E. coli
PolIII holoenzyme complex (Lahue et al., 1989, Science 245:160-164). The net result is a strand-specific mismatch repair event.
Many genetic studies performed using
E. coli
support this interpretation. For example bacteria having a mutated mutH, mutL, or mutS gene exhibit a mutator phenotype that is presumed to be the result of the increased probability of misincorporation errors leading to mutations (Demerec et al., 1957, Bact. Genet., Carnegie Inst. Wash. Yearbook 370:390-406; Miyake, 1960, Genetics 45:755-762; Siegel et al., 1967, J. Bacteriol. 94:38-47; Hill, 1970, Mutat. Res. 9:341-344). However, not all predictions arising from the
E. coli
Dam-instructed model agree with experimental results. For example, bacteria having a mutation in each of the recJ, exol, and exo VII genes do not exhibit a mutator phenotype, suggesting that other exonuclease(s) or mechanism(s) are involved in the mismatch repair process.
Homologs of the procaryotic MutS and MutL proteins have been identified in eukaryotes (Fishel et al., 1993, Cell 75:1027-1038; Prolla et al., 1994, Science 265:1091-1093; Bronner et al., 1994, Nature 368:258-261). MutH analogs appear to exist only in gram-negative bacteria.
Multiple MutS and MutL homologs have been identified in yeast and human cells which individually participate in such diverse activities as nuclear and organelle mismatch repair as well as distinct meiotic functions (Fishel et al., 1997, Curr. Opin. Genet. Dev. 7:105-113). Germ-line mutations of the human MutS and MutL Homologs, hMSH2, hMLH1, and hPMS2, have been found to be associated with the common cancer predisposition syndrome, hereditary non-polyposis colorectal cancer (HNPCC; Bronner et al., 1994, Nature 368:258-261; Fishel et al., 1993, Cell 75:1027-1038). Yeast and human MutS and MutL homologs exist primarily as heterodimeric proteins. Yeast MSH2 protein has been found to be associated with MSH3 or MSH6, and yeast MLH1 has been found to be associated with PMS1. Human hMSH2 protein has been found to be associated with hMSH3 or hMSH6 (also designated GTBP or p160 by some authors), and human hMLH1 has been found to be associated with hPMS2 (Li et al., 1995, Proc. Natl. Acad., Sci. USA 92:1950-1954; Prolla et al., 1994, Science 265:1091-1093; Drummond et al., 1995, Science 268:1909-1912; Marsischky et al., 1996, Gen. Dev. 10:407-420; Acharya et al., 1996, Proc. Natl. Acad. Sci. USA 93:13629-13634). Furthermore, MSH2/MSH3 and MSH2/MSH6 protein complexes appear to possess overlapping and redundant mismatch binding activities (Acharya et al., 1996, Proc. Natl. Acad. Sci. USA 93:13629-13634; Risinger et al., 1996, Nature Genet. 14:102-105).
Classification of MutS and MutL homologs is based on the presence in the proteins of highly conserved regions of amino acid identity. The most highly conserved region among MutS homologs includes approximately 150 amino acids which comprise a helix-turn-helix domain associated with a Walker A adenine-nucleotide and magnesium binding motif (Walker et al., 1982, EMBO J. 1:945-951). This adenine nucleotide binding domain constitutes more than 80% of the identifiable homology between MutS homologs (Fishel et al., 1997, Curr. Opin. Genet. Dev. 7:105-113). Both purified bacterial MutS homologs and purified yeast MutS homologs possess an intrinsic low-level ATPase activity (Haber et al., 1991, EMBO. J. 10:2707-2715; Chi et al., 1994, J. Biol. Chem. 269: 29993-29997; Chi et al., 1994, J. Biol. Chem. 269:29984-29992; Alani et al., 1997, Mol. Cell Biol. 17: 2436-2447). This ATPase activity is likely to be important for the function of MutS homologs, as indicated by the fact that mutation of conserved amino acid residues in the adenine nucleotide binding domain results in a dominant mutator phenotype in both bacteria and yeast (Haber et al., 1991, EMBO. J. 10:2707-2715; Wu et al., 1994, J. Bacteriol 176:5393-5400; Alani et al., 1997, Mol. Cell Biol. 17: 2436-2447). A central role for the adenine nucleotide binding domain is consistent with the ATP-dependent translocation model of mismatch repair proposed by Modrich and colleagues (Allen et al., 1997, EMBO J. 16:4467-4476).
Genetic and biochemical studies of the human mismatch repair process indicate that it is similar to bacterial mismatch repair, except that the physiologically relevant mechanism for directing strand specificity is unknown (Miller et al., 1976, Proc. Natl. Acad. Sci. USA 73:3073-3077; Glazer et al., 1987, Mol. Cell. Biol., 7:218-224; Holmes et al., 1990, Proc. Natl. Acad. Sci. USA 87:5837-5841; Thomas et al., 1991, J. Biol. Chem. 266:3744-3751; Fang et al., 1993, J. Biol. Chem. 268:11838-11844; Longley et al., 1997, J. Biol. Chem. 272:10917-10921). Purified hMSH2 protein binds mismatched nucleotides and DNA lesions (Fishel et al., 1994, Science 266:1403-1405; Fishel et al., 1994, Cancer Res. 54:5539-5542; Mello et al., 1996, Chemistry & Biology 3:579-589), and the specificity and affinity of that recognition is enhanced by association of hMSH2 with hMSH3 or hMSH6 (Drummond et al., 1995; Acharya et al., 1996, Proc. Natl. Acad. Sci. USA 93:13629-13634; Palombo et al., 1996, Curr. Biol. 6:1181-1184).
Although the ability of MutS homologs to bind to mismatched duplex DNA has been recognized (e.g. U.S. Pat. No. 5,556,750), methods of using MutS homologs in vitro have been limited by a lack of understanding regarding the properties of such homologs. A need remains for methods of binding MutS homologs and mismatched duplex DNA, which methods take advantages of the biochemical properties of such homologs.
Transgenic and Nullizygous Animals
The development of transgenic animals and nullizygous animal models has provided important new avenues for the study of specific gene functions in differentiation, embryogenesis and neoplastic development (Palmiter et al., 1986, Ann. Rev. Genet. 20:465-499). Transgenic animals frequently serve as model systems for the study of various disease states and also provide an experimental system in which to test compounds for their ability to regulate disease. Nullizygous animals are similarly useful as experimental systems for the testing of compounds useful for diagnosis, treatm
Acharya Samir
Fishel Richard A.
Gradia Scott
Akin Gump Strauss Hauer & Feld L.L.P.
Thomas Jefferson University
Zitomer Stephanie W.
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