FACTORS DETERMINING DIFFERENT PROCESSIVITY OF THERMUS-THERMOPHILUS AND T-AQUATICUS DNA-POLYMERASES IN AMPLIFICATION OF PHAGE-LAMBDA DNA

A fragment of the polymerase domain (aa 492-595) of the thermostable D NA polymerase I from Thermus thermophilus HE-8 (Tth) was replaced by t he corresponding fragment of the Escherichia coli DNA polymerase I (58 6-688). The resulting chimeric protein displayed polymerase activity w ith the optimum at 52 degrees C, which proves high homology of the str uctural-functional organization of the polymerase domains of the initi al enzymes. The same segment of the polymerase domain of Tth polymeras e was replaced by aa 490-593 of DNA polymerase I from T. aquaticus YT- I (Taq) to change 8 aa in the thumb subdomain. The resulting enzyme, n amed Ttaq polymerase, had altered processivity as compared with the in itial Tth and became in this respect analogous to Tag. At the same tim e, Ttaq retained the capacity of Tth to extend DNA containing a 3'-ter minal noncomplementary nucleotide. This implies that the different pro cessivity of Taq and Tth DNA polymerases is mainly determined by the d ifferences in the sequences of the ''thumb'' subdomain.