MECHANISM OF DNA POLYMERASE-ALPHA INHIBITION BY APHIDICOLIN

Synthetic oligonucleotides of defined sequence were used to examine the mechanism of calf thymus DNA polymerase alpha-inhibition by aphidicolin. Aphidicolin competes with each of the four dNTPs for binding to a pol alpha.DNA binary complex and thus should not be viewed as a dCTP analogue. Kinetic evidence shows that inhibition proceeds through the formation of a pol alpha.DNA.aphidicolin ternary complex, while DNase I protection experiments provide direct physical evidence. When deoxyguanosine is the next base to be replicated, K(i) = 0.2-mu-M. In contrast, the K(i) is 10-fold higher when the other dNMPs are at this position. Formation of a pol alpha.DNA.aphidicolin ternary complex did not inhibit the primase activity of the pol alpha.primase complex. Neither the rate of primer synthesis nor the size distribution of primers 2-10 nucleotides long was changed. Elongation of the primase-synthesized primers by pol a was inhibited both by ternary complex formation using exogenously added DNA and by aphidicolin alone.