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. 1996 Nov 12;93(23):12822-7.
doi: 10.1073/pnas.93.23.12822.

The Carboxyl Terminus of the Bacteriophage T4 DNA Polymerase Is Required for Holoenzyme Complex Formation

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Free PMC article

The Carboxyl Terminus of the Bacteriophage T4 DNA Polymerase Is Required for Holoenzyme Complex Formation

A J Berdis et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

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.

Figures

Figure 1
Figure 1
Carboxyl terminal alignment of the bacteriophage T4 DNA polymerase (gp43) and the bacteriophage T4 RNA polymerase binding protein (gp33). Amino acid sequence of the polypeptide used for inhibition studies is also shown.
Figure 2
Figure 2
Titration curve for the formation of the bacteriophage T4 holoenzyme complex in which the concentration of 44/62 protein was maintained at 250 nM, while the concentrations of Bio-34/62/36-mer and 45 protein were fixed at 250 nM. Streptavidin was maintained at 1 μM, while the ATP concentration was fixed at 1 mM. Before the addition of either polymerase, the steady-state rate of ATP hydrolysis was 210 nM/s. At the times indicated (arrow), either 250 nM T4 exo polymerase or 250 nM ΔC6 exo polymerase was added. (A) The ATPase activity of the 44/62 protein upon the addition of T4 exo polymerase decreased to eventually reach a limiting rate of 20 nM/s, while the ATPase activity upon the addition of ΔC6 exo polymerase did not decrease, indicating that the mutant polymerase is incapable of holoenzyme formation. (B) The ATPase activity of the 44/62 protein did not decrease upon the addition of ΔC6 exo polymerase, but did decrease upon the addition of T4 exo polymerase, indicating the specific interaction of wild-type polymerase with the 45 protein.
Figure 3
Figure 3
Holoenzyme formation and processive DNA synthesis measurements by wild-type exo polymerase alone (A), in the presence of accessory proteins (B), or by ΔC6 exo polymerase alone (C) and in the presence of accessory proteins (D). Assays were performed as described in text.
Figure 4
Figure 4
Holoenzyme formation and processive DNA synthesis measurements by wild-type exo polymerase in the presence of varying concentrations of peptide A. (A) No peptide; (B) 1 μM; (C) 5 μM; and (D) 10 μM. Assays were performed as described in text.

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