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 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.


CONCLUSIONS
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.
Kaboord
B F
BF
Pennsylvania State University, Department of Chemistry, University Park 16802, USA.
Benkovic
S J
SJ
D017122:Bacteriophage T4 / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D008969:Molecular Sequence Data; D013698:Templates, Genetic; D015534:Trans-Activators / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism*
D017931:DNA Primers; D004279:DNA, Viral; D015534:Trans-Activators; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; C074855:gene 44 protein, Enterobacteria phage T4; C074555:gene 45 protein, Enterobacteria phage T4; C088659:gp62 protein, bacteriophage T4; D004259:DNA-Directed DNA Polymerase