Biochemical characterization of interactions between DNA polymerase and single-stranded DNA-binding protein in bacteriophage RB69.
The organization and proper assembly of proteins to the primer-template junction during DNA replication is essential for accurate and processive DNA synthesis. DNA replication in RB69 (a T4-like bacteriophage) is similar to those of eukaryotes and archaea and has been a prototype for studies on DNA replication and assembly of the functional replisome. To examine protein-protein interactions at the DNA replication fork, we have established solution conditions for the formation of a discrete and homogeneous complex of RB69 DNA polymerase (gp43), primer-template DNA, and RB69 single-stranded DNA-binding protein (gp32) using equilibrium fluorescence and light scattering. We have characterized the interaction between DNA polymerase and single-stranded DNA-binding protein and measured a 60-fold increase in the overall affinity of RB69 single-stranded DNA-binding protein (SSB) for template strand DNA in the presence of DNA polymerase that is the result of specific protein-protein interactions. Our data further suggest that the cooperative binding of the RB69 DNA polymerase and SSB to the primer-template junction is a simple but functionally important means of regulatory assembly of replication proteins at the site of action. We have also shown that a functional domain of RB69 single-stranded DNA-binding protein suggested previously to be the site of RB69 DNA polymerase-SSB interactions is dispensable. The data from these studies have been used to model the RB69 DNA polymerase-SSB interaction at the primer-template junction.
Sun
Siyang
S
Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005, USA.
Shamoo
Yousif
Y
D001435:Bacteriophages / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase