Shamoo, Yousif2009-06-042009-06-042006Sun, Siyang. "Biochemical and crystallographic studies of bacteriophage RB69 DNA polymerase and single-stranded DNA binding protein interactions." (2006) Diss., Rice University. <a href="https://hdl.handle.net/1911/18982">https://hdl.handle.net/1911/18982</a>.https://hdl.handle.net/1911/18982The organization and proper assembly of proteins to the primer-template junction during DNA replication is essential for accurate and processive DNA synthesis. The DNA replication process in RB69 (a T4-like bacteriophage) is similar to the processes in eukaryotes and archaea and has been a prototype for studies on DNA replication and assembly of the functional replisome. In order to examine protein-protein interactions at the DNA replication fork, solution conditions have been established 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. The interaction between DNA polymerase and single-stranded DNA binding protein has been characterized by fluorescence titrations and results in a 60-fold increase in the overall affinity of RB69 SSB for template-strand DNA in the presence of DNA polymerase. 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. A functional domain of RB69 single-stranded DNA-binding protein previously suggested to be the site of RB69 DNA polymerase:SSB interactions has been shown to be dispensable. The data from these studies have been used to model the RB69 DNA polymerase:SSB interaction at the primer-template junction. Fusion of RB69 SSB with its cognate DNA polymerase via a short six amino acid linker increases affinity for primer-template DNA by 6-fold and increases processivity by 7-fold while maintaining fidelity. The crystal structure of this fusion protein was solved by a combination of multiwavelength anomalous diffraction and molecular replacement to 3.2 A resolution and shows that RB69 SSB is positioned proximal to the N-terminal domain of RB69 DNA polymerase near the template strand entry channel. The structural and biochemical data suggest that SSB interactions with DNA polymerase are transient and flexible, consistent with models of a dynamic replisome during elongation.200 p.application/pdfengCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.BiochemistryBiophysicsThesisTHESIS BIOCHEM. 2006 SUN