Abstract
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.
Highlights
DNA replication is an enormously complex process that requires the coordination of a number of specialized enzymatic activities involving many different proteins (1)
The results show that the gp44/62-ATP clamp loader complex binds exclusively to the C-terminal (S19C) face of the gp[45] ring
In an attempt to better understand how the gp[45] DNA sliding clamp interacts with other DNA replication proteins and DNA itself, we previously described the creation of a gp[45] mutant that contains a single cysteine residue (17)
Summary
DNA replication is an enormously complex process that requires the coordination of a number of specialized enzymatic activities involving many different proteins (1). In the phage T4 system, gp[45] loading is an ATP-dependent event that requires the action of the gp44/62 accessory proteins complex (13) Both gp[45] and, separately, template-primer DNA, stimulate the rate of ATP hydrolysis of the gp44/62 clamp loader (13, 14). In the preceding paper (18) we have used photochemical crosslinking experiments with specific mutant cysteine residues of gp[45] that had been covalently labeled with a photochemical cross-linker to probe specific interactions of the gp[45] trimer ring with the gp44/62 clamp loading machinery at various stages of the ATP-driven loading process These experiments have permitted us to begin to build some topographical details into the clamp loading reaction pathway that we (14, 17) and others (16, 19) had previously tried to define largely from a kinetic perspective. We build on these results to further examine the interaction polarity and relative positioning of the two accessory proteins subassemblies during the clamp loading process
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