ACCESSORY PROTEINS FUNCTION AS MATCHMAKERS IN THE ASSEMBLY OF THE T4 DNA-POLYMERASE HOLOENZYME

Background: During bacteriophage T4 DNA replication, the 44/62 and 45 accessory proteins combine with the DNA polymerase to form a processiv e 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 'sl iding clamp' during DNA synthesis, or whether it is required only for complex assembly. Results: To address this issue, we have stoichiometr ically assembled a processive T4 DNA polymerase holoenzyme complex, ca pable of strand-displacement synthesis, on a forked primer/template. B y titrating the 44/62 protein to substoichiometric concentrations, we have shown that it can act catalytically to load on to the primer/temp late 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 minute s, whereas the remainder have a half-life of 0.4 minutes. Precipitatio n of the protein-DNA complexes, followed by western blot analysis, ver ified that the complexes contain the DNA polymerase and 45 proteins, b ut not the 44/62 protein. Conclusion: Using physiological protein conc entrations, we have shown that the composition of the T4 protein slidi ng clamp consists solely of the 45 protein. The role of the 44/62 prot ein is that of a molecular matchmaker, in that it serves to load the 4 5 protein onto the DNA but does not remain an essential component of t he processive complex.