ABSTRACT
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.
Subject(s)
Adenoviridae/metabolism , DNA, Viral/metabolism , DNA-Binding Proteins/metabolism , Viral Proteins/metabolism , Adenosine Triphosphate/metabolism , Adenoviridae/genetics , Animals , Base Sequence , Binding Sites , Biosensing Techniques , DNA Primers/genetics , DNA Replication , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/genetics , DNA, Single-Stranded/metabolism , DNA, Viral/chemistry , DNA, Viral/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Nucleic Acid Conformation , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Deletion , Viral Proteins/chemistry , Viral Proteins/geneticsABSTRACT
In contrast to other replication systems, adenovirus DNA replication does not require a DNA helicase to unwind the double-stranded template. Elongation is dependent on the adenovirus DNA-binding protein (DBP) which has helix-destabilizing properties. DBP binds cooperatively to single-stranded DNA (ssDNA) in a non-sequence-specific manner. The crystal structure of DBP shows that the protein has a C-terminal extension that hooks on to an adjacent monomer which results in the formation of long protein chains. We show that deletion of this C-terminal arm results in a monomeric protein. The mutant binds with a greatly reduced affinity to ssDNA. The deletion mutant still stimulates initiation of DNA replication like the intact DBP. This shows that a high affinity of DBP for ssDNA is not required for initiation. On a single-stranded template, elongation is also observed in the absence of DBP. Addition of DBP or the deletion mutant has no effect on elongation, although both proteins stimulate initiation on this template. Strand displacement synthesis on a double-stranded template is only observed in the presence of DBP. The mutant, however, does not support elongation on a double-stranded template. The unwinding activity of the mutant is highly reduced compared with intact DBP. These data suggest that protein chain formation by DBP and high affinity binding to the displaced strand drive the ATP-independent unwinding of the template during adenovirus DNA replication.
Subject(s)
Adenoviridae/metabolism , DNA Replication , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Viral Proteins/chemistry , Viral Proteins/metabolism , Adenosine Triphosphate/metabolism , Adenoviridae/genetics , Animals , Baculoviridae/genetics , Base Sequence , Cell Line , DNA Primers/genetics , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/metabolism , DNA, Viral/biosynthesis , DNA, Viral/chemistry , DNA-Binding Proteins/genetics , Models, Molecular , Molecular Structure , Mutation , Nucleic Acid Conformation , Protein Conformation , Sequence Deletion , Spodoptera , Viral Proteins/geneticsABSTRACT
The cellular transcription factor nuclear factor I (NFI) stimulates adenovirus DNA replication by up to 50-fold. The NFI DNA binding domain (NFI-BD) is sufficient for stimulation and interacts with the viral DNA polymerase, thereby recruiting the precursor terminal protein-DNA polymerase complex (pTP-pol) to the origin of replication. The mechanism of DNA binding by NFI is unknown. To examine DNA binding and stimulation of adenovirus DNA replication by NFI-BD in more detail, we generated a series of deletion mutants and show that the DNA binding domain of NFI consists of two subdomains: a highly basic N-terminal domain that binds nonspecifically to DNA and a C-terminal domain that binds specifically but with very low affinity to the NFI recognition site. Both of these subdomains stimulate DNA replication, although not to the same extent as the intact DNA binding domain. The N-terminal domain has an alpha-helical structure, as shown by circular dichroism spectroscopy. The C-terminal domain interacts with the pTP-pol complex and is able to recruit the pTP-pol complex to DNA, which leads to pTP-pol-dependent stimulation of replication. The N-terminal domain also stimulates replication in a pTP-pol-dependent manner and enhances binding of pTP-pol to DNA. Since we could not detect a direct protein-protein interaction between pTP-pol and the N-terminal domain, we suggest that this domain stimulates replication by inducing structural changes in the DNA.
Subject(s)
Adenoviruses, Human/genetics , CCAAT-Enhancer-Binding Proteins , DNA Replication , DNA-Binding Proteins/genetics , Transcription Factors , Animals , Base Sequence , Binding Sites , DNA, Viral/biosynthesis , Gene Expression Regulation, Viral , Molecular Sequence Data , NFI Transcription Factors , Nuclear Proteins , Oligodeoxyribonucleotides/chemistry , Protein Folding , RNA, Messenger/genetics , Rats , Recombinant Proteins , Sequence Deletion , Structure-Activity Relationship , Virus Replication , Y-Box-Binding Protein 1ABSTRACT
The bipartite POU domain of transcription factor Oct-1 stimulates adenovirus DNA replication through an interaction with the octamer sequence present in the auxiliary origin. Employing an immobilized in vitro DNA replication system, we show that the POU domain enhances the formation of a pre-initiation complex composed of the viral precursor terminal protein-DNA polymerase (pTP-pol) complex and the origin. To investigate the mechanism of stimulation we have explored protein-protein interactions between the POU domain and the pTP-pol complex. Such an interaction could be detected using a GST-POU fusion protein bound to glutathione-agarose beads. Binding was also observed with the POU homeodomain (POUHD), albeit weaker than with the intact POU domain, but not with the POU specific subdomain. Four point mutations localized in the POUHD were analyzed for pTP-pol binding. Two of these, E22A and E30A, bound pTP-pol equally as well as the wild-type, while the other two, Q24A and E29A, were able to bind 2- to 4-fold better. These mutations are localized in the same region where the HSV transactivator VP16 binds, but did not coincide with the VP16 contacts. A direct correlation between pTP-pol binding and stimulation of DNA replication in vitro was observed for all mutants, suggesting that stimulation by the POU domain is caused by an interaction with the viral pTP-pol complex.
Subject(s)
Adenoviridae/physiology , DNA Replication , DNA, Viral/metabolism , DNA-Binding Proteins/metabolism , DNA-Directed DNA Polymerase/metabolism , Protein Structure, Tertiary , Transcription Factors/metabolism , Viral Proteins/metabolism , Virus Replication , Adenoviridae/genetics , Animals , Base Sequence , DNA Replication/drug effects , DNA, Viral/genetics , DNA-Binding Proteins/chemistry , Herpes Simplex Virus Protein Vmw65/metabolism , Host Cell Factor C1 , Macromolecular Substances , Models, Molecular , Molecular Sequence Data , NFI Transcription Factors , Octamer Transcription Factor-1 , Peptide Fragments/pharmacology , Point Mutation , Protein Binding , Rats , Recombinant Fusion Proteins/metabolism , Simplexvirus/metabolism , Transcription Factors/chemistry , Virus Replication/drug effectsABSTRACT
The POU domain is the conserved DNA binding domain of a family of gene regulatory proteins. It consists of a POU-specific domain and a POU homeodomain, connected by a variable linker region. Oct-1 is a ubiquitously expressed POU domain transcription factor. It binds to the canonical octamer sequence (ATGCAAAT) as a monomer. Here we show by chemical cross-linking and protein affinity chromatography that the Oct-1 POU domain monomers can interact in solution. This association requires both the POU homeodomain and the POU-specific domain. The interaction is transient in solution and can be stabilized by binding to the heptamer-octamer sequence in the immunoglobulin heavy-chain promoter. This correlates with cooperative DNA binding to this site. POU proteins from different subclasses, including Oct-1, Oct-2A, Oct-6, and a chimeric Oct-1 protein containing the Pit-1 POU domain, can bind cooperatively to a double binding site and form a heteromeric complex.
Subject(s)
DNA-Binding Proteins/metabolism , Immunoglobulin Heavy Chains/genetics , Promoter Regions, Genetic/genetics , Transcription Factors/metabolism , Base Sequence , Binding Sites , Chromatography , DNA-Binding Proteins/genetics , Genes, Homeobox/genetics , Glutathione Transferase/genetics , HeLa Cells , Host Cell Factor C1 , Humans , Immunoblotting , Kinetics , Macromolecular Substances , Molecular Sequence Data , Octamer Transcription Factor-1 , Recombinant Proteins/genetics , Regulatory Sequences, Nucleic Acid/genetics , Transcription Factors/genetics , Vaccinia virus/geneticsABSTRACT
POU proteins constitute a family of ubiquitous as well as cell type-specific transcription factors that share the conserved POU DNA binding domain. This domain consists of two distinct subdomains, a POU-specific domain and a POU homeodomain, that are both required for high affinity sequence-specific DNA binding. In a circular permutation assay, several POU proteins, including Oct-1, Oct-2A, Oct-6 and Pit-1, demonstrated a position dependent mobility of the protein-DNA complexes, suggesting induction of DNA bending. This was confirmed by detection of relative bend direction, using pre-bent DNA, and by enhanced ligase mediated cyclization. Bending was caused by interaction with the POU domain. By contrast, binding of the POU homeodomain did not distort the DNA structure, indicating that the POU-specific domain confers DNA bending.
