Dinucleoside polyphosphates stimulate the primer independent synthesis of poly(A) catalyzed by yeast poly(A) polymerase

Novel properties of the primer independent synthesis of poly(A), catalyzed by the yeast poly(A) polymerase are presented. The commercial enzyme fro yeast, in contrast to the enzyme from Escherichia coli, is unable to adenylate the 3'-OH end of nucleosides, nucleotides or dinucleoside polyphosphates (NpnN). In the presence of 0.05 MATP, dinucleotides (at 0.01 M) activated the enzyme velocity in the following decreasing order: Gp(4)G, 100; Gp(3)G, 82; Ap(6)A, 61; Gp(2)G, 52; Ap(4)A, 51; Ap(2)A, 41; Gp(5)G, 36; Ap(5)A, 27; Ap(3)A, 20, where 100 represents a 10-fold activation in relation to a control without effector. The velocity of the enzyme towards its substrate ATP displayed sigmoidal kinetics with a Hill coefficient (n(H)) of 1.6 and a K-m(S-0.5) value of 0.308 +/- 0.120 mM. Dinucleoside polyphosphates did not affect the maximum velocity (V-max) of the reaction, but did alter its n(H) and K-m (S-0.5) values. In the presence of 0.01 M Gp(4)G or Ap(4)A the n(H) and K-m (S-0.5) values were (1.0 and 0.063 +/- 0.012 mM) and (0.8 and 0.170 +/- 0.025 mM), respectively. With these kinetic properties, a dinucleoside polyphosphate concentration as low as 1 l M ay have a noticeable activating effect on the synthesis of poly(A) by the enzyme. These findings together with previous publications from this laboratory point to a potential relationship between dinucleoside polyphosphates and enzymes catalyzing the synthesis and/or modification of DNA or RNA.