NMR structure analysis and identification of the DNA binding site of the C-terminal domain of the Bacillus subtilis protein DnaD.

The B. subtilis protein DnaD is an essential component of the chromosome replication machinery and a global regulator of DNA architecture, as it exhibits a unique DNA remodeling activity that opens up supercoiled DNA by forming higher order nucleoprotein structures. There are no homologues of this protein in gram negative bacteria and details of its molecular structure are at present limited to the crystal structures of the N-terminal domain from B. subtilis and G. Kaustophilus, and also crystal structures of the C-terminal domain of two proteins with unknown function from S. mutans and E. faecalis (Structural Genomics projects pdb codes 2zc2 and 2i5u respectively). In this thesis, the determination of the NMR structure for the DNA binding C-terminal domain of the B. sllbtilis DnaD protein is reported. This domain is composed of five helices and an unstructured C-terminal tail, helix I-IV form a well packed hydrophobic core and helix V, which is more extensive than assumed from sequence alignments, extends away from this core fold of the protein. NMR DNA titrations, in vitro mutagenesis and in vivo complementation experiments show that a highly conserved YxxxIxxxW motif, the final helix and a portion of the mobile Cterminal region make up the DNA binding module of B. subtilis DnaD and are all essential for cell viability. Sequence alignments of DnaD alone fail to identify two key elements of this DNA binding module, the poorly conserved sequences of the final helix and the C-terminal mobile segment. Sensitive Hidden Markov Models based techniques, show that the two structural domains found in DnaD are present in B. subtilis DnaB, a primosomal protein that also participates in replication initiation. Furthermore, these two proteins share the only strong sequence motif, the highly conserved YxxxIxxxW sequence that contributes to DNA binding.