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 holoenzyme 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 exo-, is identical to wild-type exo- polymerase with respect to kcat, kpol, and dissociation constants for nucleotide and DNA substrate. However, unlike wild-type exo- polymerase, the delta C6 exo- polymerase is unable to interact with the 45 protein to form the stable holoenzyme. A synthetic polypeptide corresponding to the carboxyl terminus of the wild-type exo- polymerase was tested as an in vitro inhibitor of bacteriophage T4 DNA replication. Surprisingly, the peptide does not directly inhibit holoenzyme complex formation by disrupting the interaction of the polymerase with the 45 protein. On the contrary, the 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 for functional interactions. Data presented are discussed in terms of a model correlating the functionality of the carboxyl terminus of the polymerase for productive interactions with the 45 protein as well as in terms of the 45 protein concomitantly interacting with the 44/62 protein and polymerase.
Pennsylvania State University, Department of Chemistry, University Park 16802-6300, USA.
A J
AJ
Berdis
P
P
Soumillion
S J
SJ
Benkovic
D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000201:enzymology; D001665:Binding Sites; D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli; D007700:Kinetics; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D013379:Substrate Specificity; D014764:Viral Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D011994:Recombinant Proteins; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; D004247:DNA; D004259:DNA-Directed DNA Polymerase