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Nucleic Acids Research, 1983, Vol. 11, No. 20 7251-7260
© 1983

ENZYMOLOGY

The elongation of mismatched primers by DNA polmerasea from {alpha} calf thymus

Bernd Reckmann, Frank Grosse and Gerhard Krauss

Zentrum Biochemie, Abteilung Biophysikalische Chemie, Medizmische Hochschule Hannover, Konstaty-Gutschow-Strasse 8, 3000 Hannover, FRG

Received May 24, 1983. Revised July 13, 1983. Accepted August 13, 1983.

The ability of the 9S and 5.7S DNA polymerase {alpha} subspecies from calf thymus in elongating a mismatched primer terminus has been investigated. With poly(dA) as template, the elongation rate for (dT)8dg, (dT)8dc and (dI)10dGdT is 20-fold lower for the 9S enzyme and 5-fold lower for the 5.7S enzyme as compared to (dT)10. The presence of a second mismatch at the primer terminus redu the elongation rate further by a factor of two. Exonucleolytic excision of the mismatches can be excluded. With (dT)8dg (dT)n as primer we show, that at least five T-residues have to follow the mismatch in order to establish the elongation rate of a perfectly paired primer. The KM value for (dT)10 dG as primer is 400 nM as compared to 10 nM for (dT)10 Addition of Mn2+ increases the relative efficiency of elongation of the mismatched primers.


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