HUMAN-PLACENTA DNA POLYMERASE-ALPHA EFFICIENCY OF THE INTERACTION BETWEEN OLIGOTHYMIDYLATES OF VARYING LENGTH AND TEMPLATE SPECIFIC SITE

  • 1 March 1986
    • journal article
    • research article
    • Vol. 12  (3) , 357-368
Abstract
Modification of human placenta DNA polymerase .alpha. by (pT)2pC[Pt2+(NH3)2OH].cntdot..cntdot.(pT)7 was investigated. The linear time dependence of the enzyme activity logarithm suggested a pseudo-first order for modification. Kd value of enzyme-affinity reagent complex (0,5 .mu.M) was estimated. The enzyme inactivation by the affinity reagent and protection from inactivation in the presence of oligonucleotides of varying length were used for determining Kd values of the enzyme-ligand complexes. Oligonucleotide d(pT)2pC(pT)7 (Kd 0,15 .mu.M), d(Tp)9T (Kd 0,15 .mu.M) and [d(Tp)9]ddT (Kd 0,15 .mu.M) protected the enzyme from inactivation with equal efficiency. The protective action of oligothymidylates d(Tp)nT (where n changes from 3 to 14) strongly depended on the chain length, the Kd values diminishing from 5,3 to 0.0091 .mu.M in the geometrical progression. The addition of one link to the oligothymidylate chain resulted in 1.71-fold increase in the oligonucleotide affinity for the enzyme specific site. Such a change corresponds to Gibbs energy change of about 0,32 kcal/mole. It is supposed that the monomer units of pentadecathymidylate (at least beginning with the third one) in d(Tp)14T-enzyme complex form neither hydrogen bonds nor electrostatic linkages with the enzyme. Kd values of oligonucleotides as templates are shown to reflect quite well the true affinity of template for the enzyme. This affinity increases in the presence of 1 primer. However, the ratio of the affinity for differential oligonucleotides does not change in the presence or absence of a complementary primer.