ABSTRACT
During the assembly of the bacterial loader-dependent primosome, helicase loader proteins bind to the hexameric helicase ring, deliver it onto the oriC DNA and then dissociate from the complex. Here, to provide a better understanding of this key process, we report the crystal structure of the ~570-kDa prepriming complex between the Bacillus subtilis loader protein and the Bacillus stearothermophilus helicase, as well as the helicase-binding domain of primase with a molar ratio of 6:6:3 at 7.5 Å resolution. The overall architecture of the complex exhibits a three-layered ring conformation. Moreover, the structure combined with the proposed model suggests that the shift from the 'open-ring' to the 'open-spiral' and then the 'closed-spiral' state of the helicase ring due to the binding of single-stranded DNA may be the cause of the loader release.
Subject(s)
Bacillus/enzymology , DNA Helicases/chemistry , DNA Helicases/metabolism , DNA Replication , Adenosine Triphosphate/pharmacology , Chromatography, Gel , DNA Helicases/genetics , DNA Helicases/ultrastructure , DNA, Single-Stranded/metabolism , Escherichia coli/enzymology , Models, Molecular , Protein Structure, Tertiary , Static ElectricityABSTRACT
DNA polymerases can only synthesize nascent DNA from single-stranded DNA (ssDNA) templates. In bacteria, the unwinding of parental duplex DNA is carried out by the replicative DNA helicase (DnaB) that couples NTP hydrolysis to 5' to 3' translocation. The crystal structure of the DnaB hexamer in complex with GDP-AlF(4) and ssDNA reported here reveals that DnaB adopts a closed spiral staircase quaternary structure around an A-form ssDNA with each C-terminal domain coordinating two nucleotides of ssDNA. The structure not only provides structural insights into the translocation mechanism of superfamily IV helicases but also suggests that members of this superfamily employ a translocation mechanism that is distinct from other helicase superfamilies. We propose a hand-over-hand mechanism in which sequential hydrolysis of NTP causes a sequential 5' to 3' movement of the subunits along the helical axis of the staircase, resulting in the unwinding of two nucleotides per subunit.
Subject(s)
DnaB Helicases/chemistry , Geobacillus stearothermophilus/enzymology , Catalytic Domain , Crystallography, X-Ray , DNA Replication , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/metabolism , DnaB Helicases/metabolism , Models, Molecular , Nucleotides/metabolism , Protein Structure, TertiaryABSTRACT
The human immunodeficiency virus type 1 (HIV-1) protein Vif recruits the host E3 ubiquitin ligase, composed of cullin 5 (Cul5), Rbx2, Elongin B, and Elongin C (EloBC), to polyubiquitinate the antiviral protein APOBEC3G. Multiple regions in the C-terminal half of Vif interact with the E3 ligase. We have purified individual regions of Vif and investigated their thermodynamic contributions to the ligase assembly in vitro using isothermal titration calorimetry and fluorescence anisotropy. Our results quantify the high-affinity interactions between the Vif BC box and EloBC and between the Vif zinc finger and Cul5, as well as the modest interaction between the Vif cullin box and Cul5. Our purified Vif constructs also provide direct biochemical evidence that the Vif cullin box, containing the PPLP region, leads to the dimerization of Vif-EloBC complexes but not Cul5-Vif-EloBC complexes.
Subject(s)
HIV/metabolism , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/metabolism , vif Gene Products, Human Immunodeficiency Virus/chemistry , vif Gene Products, Human Immunodeficiency Virus/metabolism , APOBEC-3G Deaminase , Amino Acid Sequence , Cytidine Deaminase/genetics , Cytidine Deaminase/metabolism , HIV/genetics , Humans , Models, Molecular , Molecular Sequence Data , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Protein Binding , Protein Conformation , Protein Multimerization , Thermodynamics , Ubiquitin-Protein Ligases/genetics , Ubiquitination , vif Gene Products, Human Immunodeficiency Virus/geneticsABSTRACT
The complex between the DnaB helicase and the DnaG primase unwinds duplex DNA at the eubacterial replication fork and synthesizes the Okazaki RNA primers. The crystal structures of hexameric DnaB and its complex with the helicase binding domain (HBD) of DnaG reveal that within the hexamer the two domains of DnaB pack with strikingly different symmetries to form a distinct two-layered ring structure. Each of three bound HBDs stabilizes the DnaB hexamer in a conformation that may increase its processivity. Three positive, conserved electrostatic patches on the N-terminal domain of DnaB may also serve as a binding site for DNA and thereby guide the DNA to a DnaG active site.
Subject(s)
DNA Primase/chemistry , DnaB Helicases/chemistry , Geobacillus stearothermophilus/enzymology , Binding Sites , Crystallization , Crystallography, X-Ray , DNA Primase/metabolism , DNA, Bacterial/chemistry , DNA, Bacterial/metabolism , Dimerization , DnaB Helicases/metabolism , Endodeoxyribonucleases/chemistry , Endodeoxyribonucleases/metabolism , Escherichia coli/chemistry , Escherichia coli/enzymology , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Exodeoxyribonucleases/chemistry , Exodeoxyribonucleases/metabolism , Geobacillus stearothermophilus/metabolism , Image Processing, Computer-Assisted , Models, Molecular , Protein Binding , Protein Structure, Quaternary , Protein Structure, Secondary , Protein Structure, TertiaryABSTRACT
The ring-shaped hexameric DnaB helicase unwinds duplex DNA at the replication fork of eubacteria. We have solved the crystal structure of the full-length Thermus aquaticus DnaB monomer, or possibly dimer, at 2.9 A resolution. DnaB is a highly flexible two domain protein. The C-terminal domain exhibits a RecA-like core fold and contains all the conserved sequence motifs that are characteristic of the DnaB helicase family. The N-terminal domain contains an additional helical hairpin that makes it larger than previously appreciated. Several DnaB mutations that modulate its interaction with primase are found in this hairpin. The similarity in the fold of the DnaB N-terminal domain with that of the C-terminal helicase-binding domain (HBD) of the DnaG primase also includes this hairpin. Comparison of hexameric homology models of DnaB with the structure of the papillomavirus E1 helicase suggests the two helicases may function through different mechanisms despite their sharing a common ancestor.
Subject(s)
Bacterial Proteins/chemistry , DnaB Helicases/chemistry , Models, Molecular , Thermus/enzymology , Crystallography, X-Ray , Protein Structure, TertiaryABSTRACT
The Escherichia coli DEAD-box protein A (DbpA) belongs to the highly conserved superfamily-II of nucleic acid helicases that play key roles in RNA metabolism. A central question regarding helicase activity is whether the process of coupling ATP hydrolysis to nucleic acid unwinding requires an oligomeric form of the enzyme. We have investigated the structural and functional properties of DbpA by multi-angle laser light-scattering, size-exclusion chromatography, analytical ultracentrifugation, chemical cross-linking and hydrodynamic modeling. DbpA is monomeric in solution up to a concentration of 25 microM and over the temperature range of 4 degrees C to 22 degrees C. Binding of neither nucleotide (ATP or ADP) nor peptidyl transferase center (PTC) RNA, the presumed physiological RNA substrate, favor oligomerization. The hydrodynamic parameters were used together with hydrodynamic bead modeling and structural homology in conjunction with ab initio structure prediction methods to define plausible shapes of DbpA. Collectively, the results favor models where DbpA functions as an active monomer that possesses two distinct RNA binding sites, one in the helicase core domain and the other in the carboxyl-terminal domain that recognizes 23S rRNA and interacts specifically with hairpin 92 of the PTC.
