THE CARBOXYL-TERMINUS OF THE BACTERIOPHAGE-T4 DNA-POLYMERASE IS REQUIRED FOR HOLOENZYME COMPLEX-FORMATION

To further elucidate the mechanism and dynamics of bacteriophage T4 ho loenzyme formation, a mutant polymerase in which the last six carboxyl -terminal amino acids are deleted, was constructed, overexpressed, and purified to homogeneity. The mutant polymerase, designated Delta C6 e xo(-), is identical to wild-type exo(-) polymerase with respect to k(c at), k(pol), and dissociation constants for nucleotide and DNA substra te, However, unlike wild-type exo(-) polymerase, the Delta C6 exo(-) p olymerase is unable to interact with the 45 protein to form the stable holoenzyme. A synthetic polypeptide corresponding to the carboxyl ter minus of the wild-type eso polymerase was tested as an in vitro inhibi tor of bacteriophage T4 DNA replication. Surprisingly, the peptide doe s not directly inhibit holoenzyme complex formation by disrupting the interaction of the polymerase with the 35 protein. On the contrary, th e peptide appears to disrupt the interaction of the 44/62 protein with the 45 protein, suggesting that the 44/62 protein and the polymerase use the same site on the 45 protein far functional interactions, Data presented are discussed in terms of a model correlating the functional ity of the carboxyl terminus of the polymerase for productive interact ions with the 45 protein as well as in terms of the 35 protein concomi tantly interacting with the 44/62 protein and polymerase.