Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1997-07-22
2003-04-15
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C536S023100
Reexamination Certificate
active
06548277
ABSTRACT:
Throughout this application, various publications are referenced by Arabic numerals in brackets. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications are in their entirety hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
BACKGROUND OF THE INVENTION
Construction of chimaeric DNA molecules in vitro relies traditionally on two enzymatic steps catalyzed by separate protein components. Site-specific restriction endonucleases are used to generate linear DNAs with defined termini that can then be joined covalently at their ends via the action of DNA ligase.
Vaccinia DNA topoisomerase, a 314-aa virus-encoded eukaryotic type I topoisomerase [11], binds to duplex DNA and cleaves the phosphodiester backbone of one strand. The enzyme exhibits a high level of sequence specificity, akin to that of a restriction endonuclease. Cleavage occurs at a consensus pentapyrimidine element 5′-(C/T)CCTT
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in the scissile strand [12, 5, 6]. In the cleavage reaction, bond energy is conserved via the formation of a covalent adduct between the 3′ phosphate of the incised strand and a tyrosyl residue (Tyr-274) of the protein [10]. Vaccinia topoisomerase can religate the covalently held strand across the same bond originally cleaved (as occurs during DNA relaxation) or it can religate to a heterologous acceptor DNA and thereby create a recombinant molecule [7, 8].
The repertoire of DNA joining reactions catalyzed by vaccinia topoisomerase has been studied using synthetic duplex DNA substrates containing a single CCCCT cleavage site. When the substrate is configured such that the scissile bond is situated near (within 10 bp of) the 3′ end of a DNA duplex, cleavage is accompanied by spontaneous dissociation of the downstream portion of the cleaved strand [4]. The resulting topoisomerase-DNA complex, containing a 5′ single-stranded tail, can religate to an acceptor DNA if the acceptor molecule has a 5′ OH tail complementary to that of the activated donor complex. Sticky-end ligation by vaccinia toroisomerase has been demonstrated using plasmid DNA acceptors with four base overhangs created by restriction endonuclease digestion [8].
SUMMARY OF THE INVENTION
This invention provides a modified vaccinia topoisomerase enzyme containing an affinity tag which is capable of facilitating purification of protein-DNA complexes away from unbound DNA. This invention further provides a modified sequence specific topoisomerase enzyme.
This invention provides a method of ligating duplex DNAs, a method of molecular cloning of DNA, a method of synthesizing polynucleotides, and a method of gene targeting.
Lastly, this invention provides a recombinant DNA molecule composed of segments of DNA which have been joined ex vivo by the use of a sequence specific topoisomerase and which has the capacity to transform a suitable host cell comprising a DNA sequence encoding polypeptide activity.
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Shuman, S. Two classes fo DNA end-joining reactions catalyzed by vaccinia toposiomerase I. J. Biol. Chem. vol. 267(24):16755-16758, Aug. 25, 1992.*
Christiansen, et al. Eukaryotic topoisomerase I-mediated cleavage requires bipartite DNA interaction. J. Biol. Chem. vol. 268(13):9690-9701, May 5, 1993.*
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Carninci, et al., “High-efficiency full-length cDNA cloning by biotinylated CAP trapper,”Genomics, 37(3) :327-36 (1996) Academic Press, Inc. (Exhibit 21).
Carninci, et al. “High efficiency selection of full-length cDNA by improved biotinylated cap trapper,”DNA Res., 4:61-66 (1997). Universal Academy Press. (Exhibit 22).
Cheng and Shuman, “DNA strand transfer catalyzed by vaccinia topoisomerase: ligation of DNAs containing a 3′ mononucleotide overhang,”Nuc. Acids Res., 28(9):1893-8 (2000) Oxford University Press. (Exhibit 23).
Cheng and Shuman, “Recombinogenic flap ligation pathway for intrinsic repair of topoisomerase IB-induced double-strand breaks,”Mol. Cell. Biol.20(21):8059-8068 (2000) American Society for Microbiology. (Exhibit 24).
Cheng and Shuman, “Site-specific DNA transesterification by vaccinia topoisomerase: Role of specific phosphates and nucleosides,”Biochemistry38(50):16599-612 (1999) American Chemical Society. (Exhibit 25).
Cheng and Shuman, “A catalytic domain of eukaryotic DNA topoisomerase I,”J. Biol. Chem.273(19):11589-95 (1998) The American Society for Biochemistry and Molecular Biology, Inc. (Exhibit 26).
Cheng, et al., “Conservation of structure and mechanism between eukaryotic topoisomerase I and site-specific recombinases,”Cell92 (6):841-50 (1998) Cell Press. (Exhibit 27).
Cheng, et al., “Mutational analysis of 39 residues of vaccinia DNA topoisomerase identifies Lys-220, Arg-223, and Asn-228 as important for covalent catalysis,”J. Biol. Chem.272(13):8263-9 (1997) The American Society for Biochemistry and Molecular Biology, Inc. (Exhibit 28).
DiGate and Marians, “Molecular cloning and DNA sequence analysis ofEscherichia colitopoB, the gene encoding topoisomerase III,”J. Biol. Chem.264(30):17924-17930 (1989). The American Society for Biochemistry and Molecular Biology, Inc. (Exhibit 29).
Edery, et al., “An efficient strategy to isolate full-length cDNAs based on an mRNA cap retention procedure (CAPture),”Mol. Cell. Biol., 15(6):3363-3371 (1995) American Society for Microbiology. (Exhibit 30).
Ericsson, et al., “Characterization of ts 16, a temperature-sensitive mutant of vaccinia virus,”J. Virol.69(11):7072-86 (1995) American Society for Microbiology. (Exhibit 31).
Gross and Shuman, “Vaccinia virions lacking the RNA helicase nucleoside triphoshpate phosphohydrolase II are defective in early transcription,”J. Virol.70(12):8549-5 (1996) American Society for Microbiology. (Exhibit 32).
Haghighat and Sonenberg, “eIF4G dramatically enhances the binding of eIF4E to the mRNA 5′-cap structure,”J. Biol. Chem., 272(35):21677-21680 (1997) The American Society for Biochemistry and Molecular Biology, Inc. (Exhibit 33).
Haghighat et al., “The eIF4G-eIF4E complex is the target for direct cleavage by the rhinovirus 2A proteinase,”J. Virol.70:8444-8450 (1996) American Society for Microbiology. (Exhibit 34).
Henningfeld and Hecht, “A model for topoisomerase I-mediated insertions and deletions with duplex DNA substrates containing branches, nicks, and gaps,”Biochemistry34(18):6120-9 (1995) American Chemical Society. (Exhibit 35).
Invitrogen Corporation.Invitrogen Catalog, Carlsbad, California, pp. 18, 29, 43, 44, 49-52 (1998). (Exhibit 36).
Kane and Shuman, “Vaccinia virus morphogenesis is blocked by a temperature-sensitive mutation in the I7 gene that encodes a virion component,”J. Virol.67(5):2689-98 (1993) American Society for Microbiology. (Exhibit 37).
Kato, et al., “Construction of a human full-length cDNA bank,”Gene150: 243-250 (1994) Elsevier Science. (Exhibit 38).
Klemm,
Cooper & Dunham LLP
McKelvey Terry
Sandals William
Sloan-Kettering Institute for Cancer Research
White John P.
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