DNA Sequence Analysis with Modified Bacteriophage T7 

 DNA Polymerase 



Stanley Tabor, Hans E. Huber. John Rush, and Charles C. Richardson 

 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical 

 School, Boston, MA 021 15 

 (617)732-1864 



The 3' to 3' exonuclease activity of phage T7 DNA polymerase (gene 5 protein) can be 

 inactivated selectively by reactive oxygen species. The chemically modified enzyme is 

 highly processive in the presence of E. coli thioredoxin and discriminates against 

 dideoxynucleoside triphosphates (ddNTPs) only four- to sixfold. Consequently, 

 dideoxynucleotide-tenninated fragments have highly uniform radioactive intensity 

 throughout the range of a few to thousands of nucleotides in length. There is virtually 

 no background due to tenninations at pause sites or secondary-structure impediments in 

 the template. Chemically modified gene 5 protein, by virtue of having low exonuclease 

 activity, has enzymatic properties that distinguish it from native gene 5 protein. We 

 have exploited these properties to show by a chemical screen that modification of a 

 histidine residue reduces selectively the exonuclease activity. In vitro mutagenesis of 

 histidine 123, and of the neighboring residues, results in varying reduction of the 

 exonuclease activity. A deletion of 28 amino acids that encompasses His 123 eliminates 

 all exonuclease activity (< 10 " %). Incorporation of ddNTPs by T7 DNA polymerase 

 and E. coll DNA polymerase I is more efficient when Mn-* rather than Mg-* is used for 

 catalysis. SuKstituting Mn-* for Mg-* reduces the discrimination against ddNTPs 

 approximately 100-fold for DNA polymerase I and 4-fold for T7 DNA polymerase. 

 With T7 DNA polymerase and Mn'*, ddNMPs and dNMPs are incorporated at virtually 

 the same rate. Mn-* also reduces the discrimination against other analogs with 

 modifications in the furanose moiety, the base, and the phosphate hnkage. The lack of 

 discrimination against ddNTPs using the genetically modified T7 DNA polymerase and 

 Mn-* results in uniform terminations of DNA sequencing reactions, with the intensity of 

 adjacent bands on polyacrylamide gels varying in most instances by less than 10%. A 

 novel procedure that exploits the high uniformity of bands can be used for automated 

 DNA sequencing. A single reaction with a single labeled primer is carried out using 

 four different ratios of ddNTPs to dNTPs; after gel electrophoresis in a single lane, the 

 sequence at each position is determined by the relative intensity of each band. 



For more information see the following articles by S. Tabor and C. C. Richardson: 

 Proc. Natl. Acad. Sci. USA 84, 4767^771 ( 1987), J. Biol. Chem. 264, 6447-6458 

 ( 1989), and Proc. Natl. Acad. Sci. USA 86, 4076^080 (1989). 



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