Exploring the mechanism of the bacteriophage T7 single-stranded DNA binding protein gp2.5.

Single-stranded DNA binding proteins (SSB) play multivalent roles in the replication of genomic DNA. SSB binds the single-stranded (ss)DNA intermediate during replication to protect them from damage and digestions. Moreover, SSB help removes secondary structures on DNA and interacts with and stimulates activities of replicative proteins. However, the molecular basis of SSB action remains elusive. In this project, we study the function of SSB and the interplay of SSB and DNA polymerase with the DNA replication system from bacteriophage T7 due to its simplicity. T7 SSB is encoded by gene product 2.5 (gp2.5), and its replicase is comprised of gene product 5 (gp5) and host processivity factor thioredoxin. gp2.5 is a 232 residue long protein that binds to ssDNA in a monomer form but remains dimer in the solution. The 26-residue C-terminal tail plays the main role of the ssDNA binding and the interaction with other proteins. We built the protein-ssDNA force field from our previous helicase research and focused on the gp2.5 dynamics. We added an aromatic interaction term to better estimate the interactions between aromatic amino acids and DNA bases. We first explored the dimer association process and the function of electrostatics interaction of the C-terminal tail. Then we focus on the competition between the C-terminal tail and ssDNA in the binding of the OB-fold domain. We also noticed that gp2.5 shows a higher binding affinity in the poly-CG sequence than in the poly-AT sequence. In the end, we compared the structures of gp2.5, E. coli SSB, E. coli recA, and T4 gene 32 protein to find the relationship between the binding affinity and the speed of DNA synthesis.