title: Structural analyses of gp45 sliding clamp interactions during assembly of the bacteriophage T4 DNA polymerase holoenzyme. III. The Gp43 DNA polymerase binds to the same face of the sliding clamp as the clamp loader.
abstract: In the preceding paper (Latham, G. J., Bacheller, D. J., Pietroni, P. , and von Hippel, P. H. (1997) J. Biol. Chem. 272, 31677-31684), we demonstrated that the T4 gp44/62-ATP clamp loader binds to the C-terminal face of the gp45 sliding clamp. Here we extend these results by exploring the structural relationship between the gp43 polymerase and the gp45 sliding clamp. Using fluorescence intensity and polarization techniques, as well as photo-cross-linking methods, we present evidence that gp43, like gp44/62, binds to the C-terminal face of gp45. In addition, we show that g43 binds to the gp45 clamp in two distinct interaction modes, depending on the presence or absence of template-primer DNA. When template-primer DNA is present, gp43 binds tightly to gp45 to form the highly processive DNA polymerase holoenzyme. Gp43 also binds to gp45 in the absence of template-primer DNA, but this interaction is more than 100 times weaker than gp43-gp45 binding on DNA. Specific interactions between gp43 and the C-terminal face of gp45 are maintained in both modes of binding. These results underscore the pivotal role of template-primer DNA in modulating the strength of protein-protein interactions during DNA synthesis and provide additional insight into the structural requirements of the replication process.
authors.affiliation: Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, Oregon 97403-1229, USA.
authors.forename: G J
authors.initials: GJ
authors.lastname: Latham
authors.forename: D J
authors.initials: DJ
authors.lastname: Bacheller
authors.forename: P
authors.initials: P
authors.lastname: Pietroni
authors.forename: P H
authors.initials: PH
authors.lastname: von Hippel
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D046911:Macromolecular Substances; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010777:Photochemistry; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013604:T-Phages / Q000201:enzymology*; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; C102356:T7C-45 protein, Bacteriophage T4; 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; D000255:Adenosine Triphosphate; D004259:DNA-Directed DNA Polymerase