Abstract
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.
Highlights
Bound by the single-stranded DNA-binding protein (SSB) family of proteins to protect template strand DNA from nucleases and facilitate the removal of adventitious secondary structures [2]
We have characterized the interaction between DNA polymerase and singlestranded 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
We have 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
Summary
Bound by the SSB family of proteins to protect template strand DNA from nucleases and facilitate the removal of adventitious secondary structures [2]. The problem of how to efficiently bind available ssDNA is solved by the highly cooperative and nonsequence-specific interaction of the SSB family proteins for single-stranded DNA. Quantitative studies on the interaction between DNA polymerase and SSB protein have been limited because of difficulties in establishing conditions that favor formation of a discrete homogeneous complex [22]. We have found that the protein-protein cooperativity between RB69 DNA polymerase and SSB causes a roughly 60-fold increase of affinity of SSB to the adjoining single-stranded template region. These data have allowed us to construct testable models for the formation of the DNA polymerase-SSB interaction as they occur in replication forks of all organisms
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