Method for molecular cloning and polynucleotide synthesis using

Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical

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435 911, 435 914, 435 915, 4351723, C12P 1934, C12N 1509

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active

057668914

ABSTRACT:
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.

REFERENCES:
Morham, S.G., and Shuman, S. (1992) "Covalent and Noncovalent DNA Binding by Mutants of Vaccinia DNA Topoisomerase I", J. Biol. Chem. 267:15984-15992.
Shuman, S. (1991a) "Site-Specific DNA Cleavage by Vaccinia Virus DNA Topoisomerase I", J. Biol. Chem. 266:1796-1803.
Shuman, S. (1991b) "Site-Specific Interaction of Vaccinia Virus Topoisomerase I with Duplex DNA", J. Biol. Chem. 266:11372-11379.
Shuman, S. (1992a) "DNA Strand Transfer Reactions Catalyzed by Vaccinia Topoisomerase I", J. Biol. Chem. 267:8620-8627.
Shuman, S. (1992b) "Two Classes of DNA End-Joining Reactions Catalyzed by Vaccinia Topoisomerase I", J. Biol. Chem. 267:16755-16758.
Shuman, S., et al. (1988) "Characterization of Vaccinia Virus DNA Topoisomerase I Expressed in Escherichia coli", J. Biol. Chem. 263:16401-16407.
Shuman, S., et al. (1989) "Mapping the Active-Site Tyrosine of Vaccinia Virus DNA Topoisomerase I", Proc. Natl. Acad. Sci., USA 86:9793-9797.
Shuman, S. and Moss, B. (1987) "Identification of a Vaccinia Virus Gene Encoding a Type I DNA Topoisomerase", Proc. Natl. Acad. Sci., USA 84:7478-7482.
Shuman, S. and Prescott, J. (1990) "Specific DNA Cleavage and Binding of Vaccinia Virus DNA Topoisomerase I" J. Biol. Chem. 265:17826-17836.
Stivers, J.T., et al. (1994) "Vaccinia DNA Topoisomerase I: SIngle-Turnover and Steady-State Kinetic Analysis of the DNA Strand Cleavage and Ligation Reactions", Biochemistry 33:327-339.
Shuman, "Novel approach to molecular cloning and polynucleotide synthesis using vaccinia DNA topoisomerase" J. Biol. Chem. 269: 32678-32684, Dec. 1994.

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