Paracoccidioides brasiliensis infection in dogs from Western Brazilian Amazon

The objective of the study was to evaluate Paracoccidioides brasiliensis infection in urban dogs from the municipality of Monte Negro, Rondonia, Western Brazilian Amazon. The serum samples (n=126) were analyzed by indirect ELISA and the immunodiffusion test using P. brasiliensis gp43 and exoantigen as antigens, respectively. A positivity of 54.8% was observed only in the ELISA test and no statistical difference was observed in the seroprevalence in relation to age or sex. This is the first paracoccidioidomycosis survey carried out with dogs from the Western Brazilian Amazon. The higher positivity rates of P. brasiliensis infection observed in this study suggest that veterinarians must be alert to detect new cases of natural disease in dogs living in paracoccidioidomycosis endemic areas.

Crystal structure of a thermostable type B DNA polymerase from Thermococcus gorgonarius

Most known archaeal DNA polymerases belong to the type B family, which also includes the DNA replication polymerases of eukaryotes, but maintain high fidelity at extreme conditions. We describe here the 2.5 Å resolution crystal structure of a DNA polymerase from the Archaea Thermococcus gorgonarius and identify structural features of the fold and the active site that are likely responsible for its thermostable function. Comparison with the mesophilic B type DNA polymerase gp43 of the bacteriophage RB69 highlights thermophilic adaptations, which include the presence of two disulfide bonds and an enhanced electrostatic complementarity at the DNA–protein interface. In contrast to gp43, several loops in the exonuclease and thumb domains are more closely packed; this apparently blocks primer binding to the exonuclease active site. A physiological role of this “closed” conformation is unknown but may represent a polymerase mode, in contrast to an editing mode with an open exonuclease site. This archaeal B DNA polymerase structure provides a starting point for structure-based design of polymerases or ligands with applications in biotechnology and the development of antiviral or anticancer agents.

Opening of a monomer-monomer interface of the trimeric bacteriophage T4-coded GP45 sliding clamp is required for clamp loading onto DNA

The replication system of bacteriophage T4 uses a trimeric ring-shaped processivity clamp (gp45) to tether the replication polymerase (gp43) to the template-primer DNA. This ring is placed onto the DNA by an ATPase-driven clamp-loading complex (gp44/62) where it then transfers, in closed form, to the polymerase. It generally has been assumed that one of the functions of the loading machinery is to open the clamp to place it around the DNA. However, the mechanism by which this occurs has not been fully defined. In this study we design and characterize a double-mutant gp45 protein that contains pairs of cysteine residues located at each monomer-monomer interface of the trimeric clamp. This mutant protein is functionally equivalent to wild-type gp45. However, when all three monomer-monomer interfaces are tethered by covalent crosslinks formed (reversibly or irreversibly) between the cysteine pairs these closed clamps can no longer be loaded onto the DNA nor onto the polymerase, effectively eliminating processive strand-displacement DNA synthesis. Analysis of the individual steps of the clamp-loading process shows that the ATPase-dependent interactions between the clamp and the clamp loader that precede DNA binding are hyperstimulated by the covalently crosslinked ring, suggesting that binding of the closed ring induces a futile, ATP-driven, ring-opening cycle. These findings and others permit further characterization and ordering of the steps involved in the T4 clamp-loading process.

Creating a dynamic picture of the sliding clamp during T4 DNA polymerase holoenzyme assembly by using fluorescence resonance energy transfer

The coordinated assembly of the DNA polymerase (gp43), the sliding clamp (gp45), and the clamp loader (gp44/62) to form the bacteriophage T4 DNA polymerase holoenzyme is a multistep process. A partially opened toroid-shaped gp45 is loaded around DNA by gp44/62 in an ATP-dependent manner. Gp43 binds to this complex to generate the holoenzyme in which gp45 acts to topologically link gp43 to DNA, effectively increasing the processivity of DNA replication. Stopped-flow fluorescence resonance energy transfer was used to investigate the opening and closing of the gp45 ring during holoenzyme assembly. By using two site-specific mutants of gp45 along with a previously characterized gp45 mutant, we tracked changes in distances across the gp45 subunit interface through seven conformational changes associated with holoenzyme assembly. Initially, gp45 is partially open within the plane of the ring at one of the three subunit interfaces. On addition of gp44/62 and ATP, this interface of gp45 opens further in-plane through the hydrolysis of ATP. Addition of DNA and hydrolysis of ATP close gp45 in an out-of-plane conformation. The final holoenzyme is formed by the addition of gp43, which causes gp45 to close further in plane, leaving the subunit interface open slightly. This open interface of gp45 in the final holoenzyme state is proposed to interact with the C-terminal tail of gp43, providing a point of contact between gp45 and gp43. This study further defines the dynamic process of bacteriophage T4 polymerase holoenzyme assembly.