DEPENDENCE OF THE 3'-5'-EXONUCLEASE ACTIVITY IN THE KLENOW FRAGMENT OF THE ESCHERICHIA-COLI DNA-POLYMERASE-I ON THE LENGTH AND STRUCTURE OF THE OLIGONUCLEOTIDE HYDROLYZED

We derived the values of K(m) and V(max) for oligothymidylates d(pT)2-16 in the hydrolytic reaction of 3'-5'-exonuclease catalyzed by the Klenow fragment (KF) of the E. coli DNA polymerase I in the presence and absence of a poly(dA) template. We showed that, without poly(dA), the K(m) values for the oligonucleotides were but slightly affected by their length. The rate of oligothymidylate splitting increased with their increasing length, and for d(pT)16 it was about 190 times that for d(pT)2. The addition of poly(dA) failed to bring about any substantial change in the values of K(m) for d(pT)2-16, but increased the rate of hydrolysis of d(pT)2-7 by a factor of 2-17, while effectively lowering the efficiency of the hydrolysis of d(pT)8-16. The K(m) values for d(pC) 10, d(pA)10, and d(pT)10 were practically identical, while the rate of d(pC)10 hydrolysis was about 1.2 and 7.8 times those of d(pA)10 and d(pT)10, respectively. Under conditions of interaction with the template region of the enzyme, d(pC)10, d(pA)10, and d(pT)10 increased the rate of d(pT)2 hydrolysis to a different extent when that substrate was interacting with the exonuclease center of the protein. When, under the same conditions, d(pT)8, d(pT)10, and d(pT)16 were used as templates, the rate of d(pT)2 hydrolysis increased. We suggest that the exonuclease activity of KF was regulated both by the length and by the structure of the template, and that it also depended on the length and structure of the oligonucleotide hydrolyzed.