Structural analyses of gp45 sliding clamp interactions during assembly of the bacteriophage T4 DNA polymerase holoenzyme. II. The Gp44/62 clamp loader interacts with a single defined face of the sliding clamp ring.
The phage T4 gp45 sliding clamp is a ring-shaped replication accessory protein that is mounted onto DNA in an ATP-dependent manner by the gp44/62 clamp loader. In the preceding paper (Pietroni, P., Young, M. C., Latham, G. J., and von Hippel, P. H. (1997) J. Biol. Chem. 272, 31666-31676), two gp45 mutants were exploited to probe interactions of the sliding clamp ring with the gp44/62 loading machinery at various steps during the clamp loading process. In this report, these studies are extended to examine the polarity of the binding interaction between gp45 and gp44/62. Three different gp45 mutants containing a single cysteine in three topographically distinct positions were used. Several different reporter groups, including extrinsic fluorophores, a photo-cross-linker, and a biotin linker for use in a novel "streptavidin interference assay," were covalently attached to these cysteine residues. Since gp45 is a trimeric protein, these three different mutations allowed us to probe up to nine distinct local environments along the surface of the sliding clamp in the presence and absence of other replication proteins. The results show that the gp44/62-ATP clamp loader complex binds exclusively to the C-terminal (S19C) face of the gp45 ring.
Latham
G J
GJ
Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, Oregon 97403-1229, USA.
Bacheller
D J
DJ
Pietroni
P
P
von Hippel
P H
PH
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D046911:Macromolecular Substances; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D013604:T-Phages / Q000201:enzymology*; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*
D046911:Macromolecular Substances; C102356:T7C-45 protein, Bacteriophage T4; D015534:Trans-Activators; D014764:Viral Proteins; 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