Structural And Functional Research Of DnaT-ssDNA Complex Involved In DNA Replication Fork Restart

Different types of ssDNA binding domains are evolved to adapt to multiple functions and fulfil their obligations during the life cycle. There are many structural topologies that have been well studied for ssDNA binding, such as OB folds, KH domains, RNA recognition motifs (RRMs), RecA-like domain, whirly domain, and a recently defined DrpA domain. A faithful and reliable transmission of genetic material requires precise coordination and regulation of chromosome replication. This process requires the assembly of a replication complex, a primosome, at the origin. A primosome consists of several proteins, including helicase, primase, and several auxiliary proteins that are responsible for creating RNA primers on ssDNA during DNA replication. The assembly of the primosome is a fundamental step for both normal chromosomal replication and the stalled replication fork restar.DnaT is a primosomal protein that is required for the stalled replication fork restart in Escherichia coli. As an adapter, DnaT mediates the PriA-PriB-ssDNA ternary complex and the DnaB/C complex. However, the fundamental function of DnaT during PriA-dependent primosome assembly is still a black box.Here, we report the1.96A,2.08A and2.83A DnaT84-153-dT15ssDNA complex structures, which may represent two different protein and ssDNA binding states. The complex structure displays a spiral filament assembly and reveals a novel three-helix bundle single-stranded DNA binding mode. The E. coli DnaT displays a typical feature in recognising ssDNA using a base-inward fashion. DnaT84-153recognises the bases of ssDNA mainly through the conserved α2helix, while the stabilisation of the phosphodiester backbone uses L3loop and α3helix; it also combines multiple homologous domains that collaborate together to achieve the full activity level. However, both the fold and ssDNA binding mode of the DnaT84-153three-helix bundle domain are distinct from those well-known ssDNA binding domains. The2.83A DnaT84-153-dT15complex structure showed a unique ssDNA binding mode that is different from any of the known RNA binding motifs. Consequently, this structure could be the first three-helix bundle that binds to the single-stranded DNA as far as we kno w.As the complex structure exhibited a degraded DnaT coiling around the ssDNA, we analysed different lengths of DnaT fragments, including the full-length DnaT, the N-terminal-truncated DnaT84-179, and the N-and C-terminal-truncated DnaT84-153, to simulate the DnaT degradation. The biochemical analysis results revealed that both the N and C terminals of DnaT are essential to having the cooperative effect. Combined with the binding assays we arrived at a conclusion that DnaT can bind to different types of ssDNA, which is fundamental for its physiological substrate bindings.Based on binding assays and negative-staining electron microscopy results, we found that DnaT can bind to phiX174ssDNA to form nucleoprotein filaments for the first time, which indicates that DnaT might function as a scaffold protein during the PriA-dependent primosome assembly.In combination with biochemical analysis, we propose a cooperative mechanism for the binding of DnaT to ssDNA and a possible model for the assembly of PriA-PriB-ssDNA-DnaT complex that sheds light on the function of DnaT during the primosome assembly and stalled replication fork restart.