Accessory proteins function as matchmakers in the assembly of the T4 DNA polymerase holoenzyme.

Abstract:

BACKGROUND: During bacteriophage T4 DNA replication, the 44/62 and 45 accessory proteins combine with the DNA polymerase to form a processive holoenzyme complex. Formation of this complex is dependent upon ATP hydrolysis by the 44/62 protein. It is uncertain, however, whether the 44/62 protein remains with the 45 protein as part of this protein 'sliding clamp' during DNA synthesis, or whether it is required only for complex assembly. RESULTS: To address this tissue, we have stoichiometrically assembled a processive T4 DNA polymerase holoenzyme complex, capable of strand-displacement synthesis, on a forked primer/template. By titrating the 44/62 protein to substoichiometric concentrations, we have shown that it can act catalytically to load on to the primer/template the 45 protein, which, in turn, combines with the DNA polymerase to form a processive complex. Two distinct complex species are formed: most of the complexes are highly stable, with a half life of 7 minutes, whereas the remainder have a half-life of 0.4 minutes. Precipitation of the protein-DNA complexes, followed by western blot analysis, verified that the complexes contain the DNA polymerase and 45 proteins, but not the 44/62 protein. CONCLUSION: Using physiological protein concentrations, we have shown that the composition of the T4 protein sliding clamp consists solely of the 45 protein. The role of the 44/62 protein is that of a molecular matchmaker, in that it serves to load the 45 protein onto the DNA but does not remain an essential component of the processive complex.

Polymerases:

T4

Topics:

Accessory Proteins/Complexes

One line summary:

Gene product 45 is the singular component of the sliding clamp. Complex 44/62 loads gp 45 onto the DNA as a non-essential process

Status:

new topics/pols set partial results complete validated

Results:

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