2 DIFFERENT REACTIONS INVOLVED IN THE PRIMER TEMPLATE-INDEPENDENT POLYMERIZATION OF DATP AND DTTP BY TAQ DNA-POLYMERASE/

Taq and Tth DNA polymerases catalyzed polymerization of dATP and dTTP into poly d(A-T) without requiring added primer/template (Hanaki et al ., Biochem, Biophys. Res. Commun. 238, 113-118), while the Stoffel fra gment of Tag DNA polymerase and Delta Tth DNA polymerase with respecti ve deletions of ca, 290 and 250 N-terminal amino acids did not. The pr imer/template-independent polymerization appeared to proceed via two r eactions, the slow process of formation of 16-19 nt long oligo d(A-T) without primer/template and the rapid process of elongation of the oli go d(A-T) by self-priming. As the former step was more sensitive to N- ethylmaleimide than the elongation reaction, probably the formation of the oligonucleotide preceded the elongation. But when the substrates were depleted, Tag DNA polymerase degraded the high molecular weigh d( A-T) polymer to the oligomers which were resistant to the further dige stion by the 5' --> 3' exonuclease activity of Taq DNA polymerase. Pro bably, the elongation and the degradation reactions proceeded simultan eously, the former process being faster than the latter in the presenc e of enough dATP and dTTP. (C) 1998 Academic Press.