Adenovirus Single-stranded DNA-binding Protein Research Articles

Evidence That the Adenovirus Single-Stranded DNA Binding Protein Mediates the Assembly of Biomolecular Condensates to Form Viral Replication Compartments.

A common viral replication strategy is characterized by the assembly of intracellular compartments that concentrate factors needed for viral replication and simultaneously conceal the viral genome from host-defense mechanisms. Recently, various membrane-less virus-induced compartments and cellular organelles have been shown to represent biomolecular condensates (BMCs) that assemble through liquid-liquid phase separation (LLPS). In the present work, we analyze biophysical properties of intranuclear replication compartments (RCs) induced during human adenovirus (HAdV) infection. The viral ssDNA-binding protein (DBP) is a major component of RCs that contains intrinsically disordered and low complexity proline-rich regions, features shared with proteins that drive phase transitions. Using fluorescence recovery after photobleaching (FRAP) and time-lapse studies in living HAdV-infected cells, we show that DBP-positive RCs display properties of liquid BMCs, which can fuse and divide, and eventually form an intranuclear mesh with less fluid-like features. Moreover, the transient expression of DBP recapitulates the assembly and liquid-like properties of RCs in HAdV-infected cells. These results are of relevance as they indicate that DBP may be a scaffold protein for the assembly of HAdV-RCs and should contribute to future studies on the role of BMCs in virus-host cell interactions.

ATP-independent DNA unwinding by the adenovirus single-stranded DNA binding protein requires a flexible DNA binding loop

The adenovirus DNA binding protein (DBP) binds cooperatively to single-stranded (ss) DNA and stimulates both initiation and elongation of DNA replication. DBP forms protein filaments via a C-terminal arm that hooks into a neighbouring molecule. This multimerization is the driving force for ATP-independent DNA unwinding by DBP during elongation. Another conserved part of DBP forms an unstructured flexible loop that is probably directly involved in contacting DNA.By making appropriate deletion mutants that do not distort the overall DBP structure, the influence of the C-terminal arm and the flexible loop on the kinetics of ssDNA binding and on DNA replication was studied. Employing surface plasmon resonance we show that both parts of the protein are required for high affinity binding. Deletion of the C-terminal arm leads to an extremely labile DBP-ssDNA complex indicating the importance of multimerization. The flexible loop is also required for optimal stability of the DBP-ssDNA complex, providing additional evidence that this region forms part of the ssDNA-binding surface of DBP.Both deletion mutants are still able to stimulate initiation of DNA replication but are defective in supporting elongation, which may be caused by the fact that both mutants have a reduced DNA unwinding activity. Surprisingly, mixtures containing both mutants do stimulate elongation. Mixing the purified mutant proteins leads to the formation of mixed filaments that have a higher affinity for ssDNA than homogeneous mutant filaments. These results provide evidence that the C-terminal arm and the flexible loop have distinct functions in unwinding during replication. We propose the following model for ATP-independent DNA unwinding by DBP. Multimerization via the C-terminal arm is required for the formation of a protein filament that saturates the displaced strand. A high affinity of a DBP monomer for ssDNA and subsequent local destabilization of the replication fork requires the flexible loop.

Conformational change of the adenovirus DNA-binding protein induced by soaking crystals with K3UO2F5 solutions.

Soaking crystals of the C-terminal DNA-binding domain of the adenovirus single-stranded DNA-binding protein with a buffer containing K(3)UO(2)F(5) results in a 9% change of the crystallographic c axis without destruction of the crystals or appreciable loss of resolution. The crystals belong to space group P2(1)2(1)2(1) with a = 79.7, b = 75.6 and c = 60.6 A. The three-dimensional structure has been refined to 2.7 A with a crystallographic R factor of 0.206. Antiparallel chains of protein molecules running through the entire crystal are linked by uranyl ions. The relative orientation of protein monomers is flexible, even in the crystalline state, and allows changes in the packing of the protein chains.

Crystallization and Preliminary X-Ray Crystallographic Studies on the Adenovirus ssDNA Binding Protein in Complex with ssDNA

Crystals of the C-terminal domain of the adenovirus single-stranded DNA binding protein (DBP) in complex with the single-stranded oligonucleotide (dT)16 have been obtained by a batch method from material obtained by chymotryptic digest of full-length DBP. The colorless crystals grow as hexagonal prisms to a maximal size of approximately 0.85 × 0.55 × 0.55 mm. The spacegroup is P3121 with unit cell constants a = 151.5 Å and c = 124.0 Å. There are either three or four molecules of DBP per asymmetrical unit. To improve the reproducibility of crystallization, recombinant protein has been produced using a baculovirus expression system and shown to crystallize under the same conditions.

Crystal structure of the adenovirus DNA binding protein reveals a hook-on model for cooperative DNA binding.

The adenovirus single-stranded DNA binding protein (Ad DBP) is a multifunctional protein required, amongst other things, for DNA replication and transcription control. It binds to single- and double-stranded DNA, as well as to RNA, in a sequence-independent manner. Like other single-stranded DNA binding proteins, it binds ssDNA, cooperatively. We report the crystal structure, at 2.6 A resolution, of the nucleic acid binding domain. This domain is active in DNA replication. The protein contains two zinc atoms in different, novel coordinations. The zinc atoms appear to be required for the stability of the protein fold rather than being involved in direct contacts with the DNA. The crystal structure shows that the protein contains a 17 amino acid C-terminal extension which hooks onto a second molecule, thereby forming a protein chain. Deletion of this C-terminal arm reduces cooperativity in DNA binding, suggesting a hook-on model for cooperativity. Based on this structural work and mutant studies, we propose that DBP forms a protein core around which the single-stranded DNA winds.

Functional changes in temperature-sensitive mutants of the adenovirus single-stranded DNA-binding protein are accompanied by structural alterations.

Adenovirus requires the virus-encoded single-stranded DNA-binding protein (DBP) to replicate its DNA. We have previously shown (M. Tsuji, P. C. van der Vliet, and G. R. Kitchingman, J. Biol. Chem. 266:16178-16187, 1991) that the inability of three temperature-sensitive (ts) mutant DBPs (Ad2+ ND1ts23, Ad2ts111A, and Ad5ts125) to support DNA replication at the nonpermissive temperature was associated with impaired ability to bind to DNA. In this study, we examined these mutant proteins for structural alterations that might be linked to the functional changes. All three ts mutants, but not the wild-type protein, showed different proteolytic cleavage patterns before and after heating at 40 degrees C (the nonpermissive temperature), suggesting a possible conformational change during heating. The Ad2+ND1ts23 and Ad2ts111A DBPs have single amino acid changes located in a putative zinc finger subdomain (positions 282 and 280). In the presence of zinc ions, these ts mutants showed significantly increased resistance to inactivation at 40 degrees C. Surprisingly, however, the stabilizing effect of zinc was also observed with the Ad5ts125DBP, which contains a mutation located more than 100 amino acids from the zinc finger. Other related metal ions, such as cobalt, cadmium, and mercury, did not protect the ts DBPs from inactivation at 40 degrees C. These results indicate that functional changes of the ts DBPs in DNA replication and DNA binding are accompanied by structural alterations in the protein and that zinc and the metal-binding subdomain may play an important role in the structure and/or function of the DBP.

Temperature-sensitive mutants of adenovirus single-stranded DNA-binding protein. Inability to support DNA replication is associated with an altered DNA-binding activity of the protein

The adenovirus single-stranded DNA-binding protein (DBP) is an essential factor in viral DNA replication. Three temperature-sensitive (ts) adenoviruses (Ad2+ND1ts23, Ad2ts111A, and Ad5ts125) are known to have single amino acid substitutions in their DBPs that result in defective DNA replication at the nonpermissive temperature. To elucidate the mechanism(s) involved in the ts phenotype, we purified the three mutant DBPs and studied their DNA-binding properties and their ability to support DNA replication in an in vitro system. The results confirm that the three ts DBPs were incapable of supporting DNA replication at the nonpermissive temperature (40 degrees C). The defect was found at both the initiation and elongation steps of DNA replication. The 2-fold stimulation of pTP.dCMP formation by the DBP was lost by prior heating of the ts DBPs. The pronounced effect of the DBP on the early elongation process was severely diminished, but not abolished, by prior heating to 40 degrees C. The functional change at 40 degrees C was irreversible, as the ts DBPs preincubated at 40 degrees C were no longer active when assayed at 30 degrees C. Upon heating to 40 degrees C, all three ts DBPs lost their ability to bind to oligonucleotides, although they still retained some binding activity for large single-stranded DNAs such as M13 DNA. Thus, the inability of these three ts DBPs to support DNA replication is attributable to their altered DNA-binding properties.

Localization of Adenovirus-encoded DNA Replication Proteins in the Nucleus by Immunogold Electron Microscopy

The distribution of three adenovirus-encoded DNA replication proteins in the nucleus of human 293 cells was studied by immunogold electron microscopy. The infected nuclei contained four morphologically distinct inclusions. They were highly electron-dense granules (type I), compact fibrogranular masses of medium electron density (type II), filamentous masses of low electron density (type III) and large polygonal crystals (type IV). In immunogold labelling studies, antibodies to the adenovirus single-stranded DNA-binding protein (DBP) and antibodies to single-stranded DNA showed extensive binding to the type III inclusions. The antibodies to the adenovirus DNA polymerase (AdPol) and terminal protein (TP) predominantly labelled type II inclusions. Double immunogold labelling studies detected low levels of AdPol and TP in type III inclusions and DBP in type II inclusions. The selective distribution of DNA replication proteins suggests that the type II and III inclusions represent two functionally different entities that may be involved in two different aspects of adenovirus DNA replication, i.e. chain initiation and elongation.

Protein kinases associated with the adenovirus single-stranded DNA-binding protein.

Protein kinase activities copurifying with the 72000-Mr DNA-binding protein of adenovirus on DNA-cellulose chromatography and gel filtration in acrylamide/agarose have been partially characterized and purified. One of these kinases was found to phosphorylate efficiently the viral DNA-binding protein in vitro and to be stimulated severalfold by the addition of histones, protamine, or polyamines. The kinase does not, however, phosphorylate histones, protamine, casein, or phosvitin. A second protein kinase was also recovered from single-stranded DNA-cellulose which is able to phosphorylate the 72000-Mr DNA-binding protein, but which is inhibited by the addition of histones. Phosphorylation in vitro of the 72000-Mr DNA-binding protein from the ts125 mutants of adenovirus by the histone-stimulated protein kinase was found to be thermosensitive.